Multi-step seperation process

ABSTRACT

The present invention provides a chromatographic separation process for recovering a polyunsaturated fatty acid (PUFA) product from a feed mixture, which comprises: (a) purifying the feed mixture in a first chromatographic separation step using an eluent a mixture of water and a first organic solvent, to obtain an intermediate product; and (b) purifying the intermediate product in a second chromatographic separation step using as eluent a mixture of water and a second organic solvent, to obtain the PUFA product, wherein the second organic solvent is different from the first organic solvent and has a polarity index which differs from the polarity index of the first organic solvent by between 0.1 and 2.0, wherein the PUFA product is other than alpha-linolenic acid (ALA), gamma-linolenic acid (GLA), linoleic acid, an ALA mono- di- or triglyceride, a GLA mono- di- or triglyceride, a linoleic acid mono- di- or triglyceride, an ALA C 1 -C 4  alkyl ester, a GLA C 1 -C 4  alkyl ester or a linoleic acid C 1 -C 4  alkyl ester or a mixture thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/759,764, filed Jul. 8, 2015, which is theNational Phase entry of International Application No. PCT/GB2014/050054,filed Jan. 9, 2014, which claims priority of Great Britain PatentApplication No. 1300354.6, filed Jan. 9, 2013, and claims the benefit ofU.S. Provisional Patent Application No. 61/750,389, filed Jan. 9, 2013.The contents of these related applications are incorporated by referenceherein in their entireties.

DESCRIPTION

The present invention relates to an improved chromatographic separationprocess for purifying polyunsaturated fatty acids (PUFAs) andderivatives thereof. In particular, the present invention relates to animproved chromatographic separation process which employs a mixedsolvent system.

BACKGROUND OF THE INVENTION

Fatty acids, in particular PUFAs, and their derivatives are precursorsfor biologically important molecules, which play an important role inthe regulation of biological functions such as platelet aggregation,inflammation and immunological responses. Thus, PUFAs and theirderivatives may be therapeutically useful in treating a wide range ofpathological conditions including CNS conditions; neuropathies,including diabetic neuropathy; cardiovascular diseases; general immunesystem and inflammatory conditions, including inflammatory skindiseases.

PUFAs are found in natural raw materials, such as vegetable oils andmarine oils. Such PUFAs are, however, frequently present in such oils inadmixture with saturated fatty acids and numerous other impurities.PUFAs should therefore desirably be purified before nutritional orpharmaceutical uses.

Unfortunately, PUFAs are extremely fragile. Thus, when heated in thepresence of oxygen, they are prone to isomerization, peroxidation andoligomerization. The fractionation and purification of PUFA products toprepare pure fatty acids is therefore difficult. Distillation, evenunder vacuum, can lead to non-acceptable product degradation.

Chromatographic separation techniques are well known to those of skillin the art. Chromatographic separation techniques involving stationarybed systems and simulated or actual moving bed systems are both familiarto one of skill in the art.

In a conventional stationary bed chromatographic system, a mixture whosecomponents are to be separated percolates through a container. Thecontainer is generally cylindrical, and is typically referred to as thecolumn. The column contains a packing of a porous material (generallycalled the stationary phase) exhibiting a high permeability to fluids.The percolation velocity of each component of the mixture depends on thephysical properties of that component so that the components exit fromthe column successively and selectively. Thus, some of the componentstend to fix strongly to the stationary phase and thus will percolateslowly, whereas others tend to fix weakly and exit from the column morequickly. Many different stationary bed chromatographic systems have beenproposed and are used for both analytical and industrial productionpurposes.

Simulated and actual moving bed chromatography are known techniques,familiar to those of skill in the art. The principle of operationinvolves countercurrent movement of a liquid eluent phase and a solidadsorbent phase. This operation allows minimal usage of solvent makingthe process economically viable. Such separation technology has foundseveral applications in diverse areas, including hydrocarbons,industrial chemicals, oils, sugars and APIs.

Thus, a simulated moving bed chromatography apparatus consists of anumber of individual columns containing adsorbent which are connectedtogether in series. Eluent is passed through the columns in a firstdirection. The injection points of the feedstock and the eluent, and theseparated component collection points in the system, are periodicallyshifted by means of a series of valves. The overall effect is tosimulate the operation of a single column containing a moving bed of thesolid adsorbent, the solid adsorbent moving in a countercurrentdirection to the flow of eluent. Thus, a simulated moving bed systemconsists of columns which, as in a conventional stationary bed system,contain stationary beds of solid adsorbent through which eluent ispassed, but in a simulated moving bed system the operation is such as tosimulate a continuous countercurrent moving bed.

A typical simulated moving bed chromatography apparatus is illustratedwith reference to FIG. 1. The concept of a simulated or actual movingbed chromatographic separation process is explained by considering avertical chromatographic column containing stationary phase S dividedinto sections, more precisely into four superimposed sub-zones I, II,III and IV going from the bottom to the top of the column. The eluent isintroduced at the bottom at IE by means of a pump P. The mixture of thecomponents A and B which are to be separated is introduced at IA+Bbetween sub-zone II and sub-zone III. An extract containing mainly B iscollected at SB between sub-zone I and sub-zone II, and a raffinatecontaining mainly A is collected at SA between sub-zone III and sub-zoneIV.

In the case of a simulated moving bed system, a simulated downwardmovement of the stationary phase S is caused by movement of theintroduction and collection points relative to the solid phase. In thecase of an actual moving bed system, simulated downward movement of thestationary phase S is caused by movement of the various chromatographiccolumns relative to the introduction and collection points. In FIG. 1,eluent flows upward and mixture A+B is injected between sub-zone II andsub-zone III. The components will move according to theirchromatographic interactions with the stationary phase, for exampleadsorption on a porous medium. The component B that exhibits strongeraffinity to the stationary phase (the slower running component) will bemore slowly entrained by the eluent and will follow it with delay. Thecomponent A that exhibits the weaker affinity to the stationary phase(the faster running component) will be easily entrained by the eluent.If the right set of parameters, especially the flow rate in eachsub-zone, are correctly estimated and controlled, the component Aexhibiting the weaker affinity to the stationary phase will be collectedbetween sub-zone III and sub-zone IV as a raffinate and the component Bexhibiting the stronger affinity to the stationary phase will becollected between sub-zone I and sub-zone II as an extract.

It will therefore be appreciated that the conventional simulated movingbed system schematically illustrated in FIG. 1 is limited to binaryfractionation.

Processes and equipment for simulated moving bed chromatography aredescribed in several patents, including U.S. Pat. No. 2,985,589, U.S.Pat. No. 3,696,107, U.S. Pat. No. 3,706,812, U.S. Pat. No. 3,761,533,FR-A-2103302, FR-A-2651148 and FR-A-2651149, the entirety of which areincorporated herein by reference. The topic is also dealt with at lengthin “Preparative and Production Scale Chromatography”, edited by Ganetsosand Barker, Marcel Dekker Inc, New York, 1993, the entirety of which isincorporated herein by reference.

An actual moving bed system is similar in operation to a simulatedmoving bed system. However, rather than shifting the injection points ofthe feed mixture and the eluent, and the separated component collectionpoints by means of a system of valves, instead a series of adsorptionunits (i.e. columns) are physically moved relative to the feed anddrawoff points. Again, operation is such as to simulate a continuouscountercurrent moving bed.

Processes and equipment for actual moving bed chromatography aredescribed in several patents, including U.S. Pat. No. 6,979,402, U.S.Pat. No. 5,069,883 and U.S. Pat. No. 4,764,276, the entirety of whichare incorporated herein by reference.

Purification of PUFA products is particularly challenging. Thus, manysuitable feedstocks for preparing PUFA products are extremely complexmixtures containing a large number of different components with verysimilar retention times in chromatography apparatuses. It is thereforevery difficult to separate certain PUFAs from such feedstocks. However,a high degree of purity of PUFA products is required, particularly forpharmaceutical and nutraceutical applications. Historically, therefore,distillation has been used when high purity PUFA products are required.There are, however, significant drawbacks to using distillation as aseparation technique for delicate PUFAs as discussed above.

Published international patent application WO-A-2011/080503, theentirety of which is incorporated herein by reference, discloses an SMBseparation process for recovering a PUFA product from a feed mixtureefficiently and in very high purity. It has been found, however, that itcan be difficult to remove C18 fatty acids, in particularalpha-linolenic acid (ALA) and/or gamma-linolenic acid (GLA), from feedmixtures efficiently without using large volumes of aqueous alcoholsolvents. Efficient removal of C18 fatty acids is advantageous sincemany specifications for pharmaceutical and dietary oils require a lowcontent of these fatty acids. For example, certain oil specificationsfor use in Japan require an ALA content of less than 1 wt %.

Accordingly, there is a need for a chromatographic separation processwhich can efficiently recover a PUFA product from a feed mixture whilstminimising the amount of C18 fatty acids, for example ALA and/or GLA,present in the resultant product.

SUMMARY OF THE INVENTION

It has now been surprisingly found that a PUFA product with low levelsof C18 fatty acids, for example ALA and/or GLA, can be effectivelypurified from commercially available feedstocks such as fish oils byusing a mixed solvent system.

The present invention therefore provides a chromatographic separationprocess for recovering a polyunsaturated fatty acid (PUFA) product froma feed mixture, which comprises:

-   -   (a) purifying the feed mixture in a first chromatographic        separation step using as eluent a mixture of water and a first        organic solvent, to obtain an intermediate product; and    -   (b) purifying the intermediate product in a second        chromatographic separation step using as eluent a mixture of        water and a second organic solvent, to obtain the PUFA product,    -   wherein the second organic solvent is different from the first        organic solvent and has a polarity index which differs from the        polarity index of the first organic solvent by between 0.1 and        2.0,        wherein the PUFA product is not alpha-linolenic acid (ALA),        gamma-linolenic acid (GLA), linoleic acid, an ALA mono- di- or        triglyceride, a GLA mono- di- or triglyceride, a linoleic acid        mono, di- or triglyceride, an ALA C₁-C₄ alkyl ester, a GLA C₁-C₄        alkyl ester or a linoleic acid C₁-C₄ alkyl ester or a mixture        thereof.

Also provided is a PUFA product obtainable by the process of the presentinvention.

Also provided is a composition comprising a PUFA product obtainable bythe process of the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the basic principles of a simulated or actual movingbed process for separating a binary mixture.

FIG. 2 illustrates a chromatographic separation step, which comprisestwo simulated or actual moving bed processes, to separate EPA fromfaster and slower running impurities (i.e. more polar and less polarimpurities).

FIG. 3 illustrates a chromatographic separation step, which comprisestwo simulated or actual moving bed processes, to separate DHA fromfaster and slower running impurities (i.e. more polar and less polarimpurities).

FIG. 4 illustrates a chromatographic separation step, which comprisestwo simulated or actual moving bed processes, to separate EPA fromfaster and slower running impurities (i.e. more polar and less polarimpurities).

FIG. 5 illustrates a chromatographic separation step, which comprisestwo simulated or actual moving bed processes, to separate DHA fromfaster and slower running impurities (i.e. more polar and less polarimpurities).

FIG. 6 illustrates a chromatographic separation step, which comprisestwo simulated or actual moving bed processes, to separate EPA fromfaster and slower running impurities (i.e. more polar and less polarimpurities).

FIG. 7 illustrates a chromatographic separation step, which comprisestwo simulated or actual moving bed processes, to separate DHA fromfaster and slower running impurities (i.e. more polar and less polarimpurities).

FIG. 8 illustrates a chromatographic separation step, which comprisestwo simulated or actual moving bed processes, to separate EPA fromfaster and slower running impurities (i.e. more polar and less polarimpurities).

FIG. 9 illustrates a chromatographic separation step, which comprisestwo simulated or actual moving bed processes, to separate EPA fromfaster and slower running impurities (i.e. more polar and less polarimpurities).

FIG. 10 illustrates three ways in which a chromatographic separationstep which comprises two simulated or actual moving bed processes may becarried out.

FIG. 11 illustrates a chromatographic separation step to separate EPAfrom faster and slower running impurities (i.e. more polar and lesspolar impurities).

FIG. 12 shows a GC-FAMES trace of an intermediate product produced bythe first separation step of the process of the present invention wheremethanol is used as first organic solvent.

FIG. 13 shows a GC-FAMES trace of a PUFA product produced by the secondseparation step of the process of the present invention whereacetonitrile is used as second organic solvent.

FIG. 14 shows a GC-FAMES trace of an intermediate product produced bythe first separation step of the process of the present invention whereacetonitrile is used as first organic solvent.

FIG. 15 shows a GC-FAMES trace of a PUFA product produced by the secondseparation step of the process of the present invention where methanolis used as second organic solvent.

FIG. 16 shows a GC-FAMES trace of a typical feed mixture, which contains55% wt % EPA ethyl ester.

DETAILED DESCRIPTION OF THE INVENTION

In its most general sense, the present invention provides achromatographic separation process for recovering a polyunsaturatedfatty acid (PUFA) product from a feed mixture, which comprises:

-   -   (a) purifying the feed mixture in a first chromatographic        separation step using as eluent a mixture of water and a first        organic solvent, to obtain an intermediate product; and    -   (b) purifying the intermediate product in a second        chromatographic separation step using as eluent a mixture of        water and a second organic solvent, to obtain the PUFA product,        wherein the second organic solvent is different from the first        organic solvent and has a polarity index which differs from the        polarity index of the first organic solvent by between 0.1 and        2.0.

As used herein, the term “PUFA product” refers to a product comprisingone or more polyunsaturated fatty acids (PUFAs), and/or derivativesthereof, typically of nutritional or pharmaceutical significance.Typically, the PUFA product is a single PUFA or derivative thereof.Alternatively, the PUFA product is a mixture of two or more PUFAs orderivatives thereof.

The term “polyunsaturated fatty acid” (PUFA) refers to fatty acids thatcontain more than one double bond. Such PUFAs are well known to theperson skilled in the art. As used herein, a PUFA derivative is a PUFAin the form of a mono-, di- or tri-glyceride, ester, phospholipid,amide, lactone, or salt. Mono-, di- and triglycerides and esters arepreferred. Triglycerides and esters are more preferred. Esters are evenmore preferred. Esters are typically alkyl esters, preferably C₁-C₆alkyl esters, more preferably C₁-C₄ alkyl esters. Examples of estersinclude methyl and ethyl esters. Ethyl esters are most preferred.

Typically, the PUFA product is at least one ω-3 or ω-6 PUFA or aderivative thereof, preferably at least one ω-3 PUFA or a derivativethereof.

Examples of ω-3 PUFAs include eicosatrienoic acid (ETE),eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA),docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). EPA, DPA andDHA are preferred. EPA and DHA are most preferred.

Examples of ω-6 PUFAs include eicosadienoic acid, dihomo-gamma-linolenicacid (DGLA), arachidonic acid (ARA), docosadienoic acid, adrenic acidand docosapentaenoic (ω-6) acid. ARA and DGLA are preferred.

Preferably, the PUFA product is EPA, DHA, a derivative thereof ormixtures thereof. Typical derivatives include EPA and DHA mono-, di- andtriglycerides and EPA and DHA esters, preferably alkyl esters such asC₁-C₄ alkyl esters.

More preferably, the PUFA product is EPA, DHA, or a derivative thereof.Typical derivatives include EPA and DHA mono-, di- and triglycerides andEPA and DHA esters, preferably alkyl esters such as C₁-C₄ alkyl esters.

Most preferably, the PUFA product is eicosapentaenoic acid (EPA),docosahexaenoic acid (DHA), EPA triglycerides, DHA triglycerides, EPAethyl ester or DHA ethyl ester.

Particularly preferably, the PUFA product is EPA, DHA, EPA ethyl esteror DHA ethyl ester.

In one embodiment, the PUFA product is EPA and/or EPA ethyl ester (EE)

In another embodiment, the PUFA product is DHA and/or DHA ethyl ester(EE).

In a yet further embodiment, the PUFA product is a mixture of EPA andDHA and/or EPA EE and DHA EE.

In a most preferred embodiment, the PUFA product obtained in the secondseparation step is EPA or an EPA derivative, for example EPA ethylester, and is obtained at a purity greater than 90 wt %, preferablygreater than 95 wt %, more preferably greater than 97 wt %, even morepreferably greater than 98 wt %, still more preferably greater than 98.4wt %. Preferably, the PUFA product obtained in the second separationstep is EPA or an EPA derivative, for example EPA ethyl ester, and isobtained at a purity between 98 and 99.5 wt %.

Typically, in addition to said PUFA product, an additional secondaryPUFA product is collected in the chromatographic separation process ofthe invention. Preferably, the PUFA product is EPA or a derivativethereof and the additional secondary PUFA product is DHA or a derivativethereof.

In a further embodiment of the invention, the process is configured tocollect a PUFA product which is a concentrated mixture of EPA and DHA orderivatives thereof. Thus, a feed mixture is used which contains EPA,DHA, components which are more polar than EPA and DHA, and componentswhich are less polar than EPA and DHA.

Typically, the PUFA product contains less than 1 wt % of alpha-linolenicacid (ALA), ALA mono-, di- and triglyceride and ALA C₁-C₄ alkyl esterimpurities. More typically, the PUFA product contains less than 1 wt %of impurities which are ALA and derivatives thereof. Typical ALAderivatives are as defined above for PUFA derivatives.

Typically, the PUFA product contains less than 1 wt % of gamma-linolenicacid (GLA), GLA mono-, di- and triglyceride and GLA C₁-C₄ alkyl esterimpurities. More typically, the PUFA product contains less than 1 wt %of impurities which are GLA and derivatives thereof. Typical GLAderivatives are as defined above for PUFA derivatives.

Typically, the PUFA product contains less than 1 wt % of C18 fatty acidimpurities, C18 fatty acid mono-, di- and triglyceride impurities andC18 fatty acid alkyl ester impurities. More typically, the PUFA productcontains less than 1 wt % of impurities which are C18 fatty acids andderivatives thereof. Typical C18 fatty acid derivatives are as definedabove for PUFA derivatives. As used herein, a C18 fatty acid is a C18aliphatic monocarboxylic acid having a straight or branched hydrocarbonchain. Typical C18 fatty acids include stearic acid (C18:0), oleic acid(C18:1n9), vaccenic acid (C18:1n7), linoleic acid (C18:2n6),gamma-linolenic acid/GLA (C18:3n6), alpha-linolenic acid/ALA (C18:3n3)and stearidonic acid/SDA (C18:4n3).

For the avoidance of doubt, in these embodiments the maximum amount ofall of the specified impurities is 1 wt %.

As explained above, typically the amount of the above-mentionedimpurities in the PUFA product is less than 1 wt %. Preferably, theamount of the above-mentioned impurities is less than 0.5 wt %, morepreferably less than 0.25 wt %, even more preferably less than 0.1 wt %,yet more preferably less than 0.05 wt %, yet more preferably less than0.01 wt %, yet more preferably less than 0.001 wt %, yet more preferablyless than 0.0001 wt %, yet more preferably less than 0.00001 wt %.

In certain preferred embodiments, the PUFA product is substantially freeof the above-mentioned impurities.

The PUFA product is not ALA, GLA, linoleic acid, an ALA mono- di- ortriglyceride, a GLA mono- di- or triglyceride, a linoleic acid mono, di-or triglyceride, an ALA C₁-C₄ alkyl ester, a GLA C₁-C₄ alkyl ester or alinoleic acid C₁-C₄ alkyl ester or a mixture thereof. Typically, thePUFA product is not ALA, GLA, linoleic acid, or a derivative or mixturesthereof. Typical ALA, GLA and linoleic acid derivatives are as definedabove for PUFA derivatives.

Typically, the PUFA product is not a C18 PUFA, a C18 PUFA mono-, di- ortriglyceride, or a C18 PUFA alkyl ester. Thus, the present inventionprovides a chromatographic separation process for recovering apolyunsaturated fatty acid (PUFA) product from a feed mixture, whichcomprises:

(a) purifying the feed mixture in a first chromatographic separationstep using as eluent a mixture of water and a first organic solvent, toobtain an intermediate product; and

(b) purifying the intermediate product in a second chromatographicseparation step using as eluent a mixture of water and a second organicsolvent, to obtain the PUFA product, wherein the second organic solventis different from the first organic solvent and has a polarity indexwhich differs from the polarity index of the first organic solvent bybetween 0.1 and 2.0,

wherein the PUFA product is other than a C18 PUFA, a C18 PUFA mono-, di-or triglyceride, or a C18 PUFA alkyl ester.

More typically, the PUFA product is not a C18 PUFA or a C18 PUFAderivative. Typical C18 PUFAs include linoleic acid (C18:2n6), GLA(C18:3n6), and ALA (C18:3n3).

Suitable feed mixtures for separating by the process of the presentinvention may be obtained from natural sources including vegetable andanimal oils and fats, and from synthetic sources including oils obtainedfrom genetically modified plants, animals and micro organisms includingyeasts. Examples include fish oils, algal and microalgal oils and plantoils, for example borage oil, Echium oil and evening primrose oil. Inone embodiment, the feed mixture is a fish oil. In another embodiment,the feed mixture is an algal oil. Algal oils are particularly suitablewhen the desired PUFA product is EPA and/or DHA. Genetically modifiedyeast is particularly suitable when the desired PUFA product is EPA.

In a particularly preferred embodiment the feed mixture is a fish oil orfish-oil derived feedstock. It has advantageously been found that when afish-oil or fish-oil derived feed stock is used, an EPA or EPA ethylester PUFA product can be produced by the process of the presentinvention in greater than 90% purity, preferably greater than 95%purity, more preferably greater than 97% purity, even more preferablygreater than 98 wt %, still more preferably greater than 98.4 wt %, forexample between 98 and 99.5 wt %.

The feed mixture may undergo chemical treatment before fractionation bythe process of the invention. For example, it may undergo glyceridetransesterification or glyceride hydrolysis followed in certain cases byselective processes such as crystallisation, molecular distillation,urea fractionation, extraction with silver nitrate or other metal saltsolutions, iodolactonisation or supercritical fluid fractionation.Alternatively, a feed mixture may be used directly with no initialtreatment step.

The feed mixtures typically contain the PUFA product and at least onemore polar component and at least one less polar component. The lesspolar components have a stronger adherence to the adsorbent used in theprocess of the present invention than does the PUFA product. Duringoperation, such less polar components typically move with the solidadsorbent phase in preference to the liquid eluent phase. The more polarcomponents have a weaker adherence to the adsorbent used in the processof the present invention than does the PUFA product. During operation,such more polar components typically move with the liquid eluent phasein preference to the solid adsorbent phase. In general, more polarcomponents will be separated into a raffinate stream, and less polarcomponents will be separated into an extract stream.

The feed mixture typically contains the PUFA product and at least oneC18 fatty acid impurity as defined above. Thus, more typically the feedmixture contains the PUFA product and at least one C18 fatty acid and/orderivative thereof. Typical C18 fatty acid derivatives are as definedabove for PUFA derivatives. Preferably, the feed mixture contains thePUFA product and at least one C18 fatty acid impurity chosen fromstearic acid (C18:0), oleic acid (C18:1n9), vaccenic acid (C18:1n7),linoleic acid (C18:2n6), gamma-linolenic acid/GLA (C18:3n6),alpha-linolenic acid (C18:3n3) and stearidonic acid/SDA (C18:4n3) andderivatives thereof.

Preferably, the feed mixture comprises (i) the PUFA product, and/or amono-, di- or triglyceride of the PUFA product and/or a C₁-C₄ alkylester of the PUFA product, and (ii) ALA and/or a mono-, di- ortriglyceride of ALA and/or a C₁-C₄ alkyl ester of ALA.

Preferably, the feed mixture comprises (i) the PUFA product, and/or amono-, di- or triglyceride of the PUFA product and/or a C₁-C₄ alkylester of the PUFA product, and (ii) GLA and/or a mono-, di- ortriglyceride of GLA and/or a C₁-C₄ alkyl ester of GLA.

More preferably, the feed mixture comprises (i) the PUFA product, and/ora mono-, di- or triglyceride of the PUFA product and/or a C₁-C₄ alkylester of the PUFA product, and (ii) ALA and/or GLA and/or a mono-, di-or triglyceride of ALA and/or a mono-, di- or triglyceride of GLA and/ora C₁-C₄ alkyl ester of ALA and/or a C₁-C₄ alkyl ester of GLA.

In embodiments where the PUFA product contains less than 1 wt % of theabove-specified C18 fatty acid impurities, the feed mixture typicallycontains the specified C18 fatty acid impurities. Thus, it is aparticular advantage of the present invention that the amount of C18fatty acid impurities present in a feed mixture can be reduced to a lowlevel by the process of the present invention. For example, where thePUFA product contains less than 1 wt % of ALA, ALA mono-, di- andtriglycerides and ALA C₁-C₄ alkyl esters, the feed mixture typicallycontains ALA, ALA mono-, di- and triglycerides and/or ALA C₁-C₄ alkylesters. Where the PUFA product contains less than 1 wt % of GLA, GLAmono-, di- and triglycerides and GLA C₁-C₄ alkyl esters, the feedmixture typically contains GLA, GLA mono-, di- and triglycerides and/orGLA C₁-C₄ alkyl esters. Where the PUFA product contains less than 1 wt %of C18 fatty acids, C18 fatty acid mono-, di- and triglycerides and C18fatty acid alkyl esters, the feed mixture typically contains C18 fattyacids, C18 fatty acid mono-, di- and triglycerides and/or C18 fatty acidalkyl esters.

Examples of the more and less polar components include (1) othercompounds occurring in natural oils (e.g. marine oils or vegetableoils), (2) byproducts formed during storage, refining and previousconcentration steps and (3) contaminants from solvents or reagents whichare utilized during previous concentration or purification steps.

Examples of (1) include other unwanted PUFAs; saturated fatty acids;sterols, for example cholesterol; vitamins; and environmentalpollutants, such as polychlorobiphenyl (PCB), polyaromatic hydrocarbon(PAH) pesticides, chlorinated pesticides, dioxines and heavy metals.PCB, PAH, dioxines and chlorinated pesticides are all highly non-polarcomponents.

Examples of (2) include isomers and oxidation or decomposition productsfrom the PUFA product, for instance, auto-oxidation polymeric productsof fatty acids or their derivatives.

Examples of (3) include urea which may be added to remove saturated ormono-unsaturated fatty acids from the feed mixture.

Preferably, the feed mixture is a PUFA-containing marine oil (e.g. afish oil), more preferably a marine oil (e.g. a fish oil) comprising EPAand/or DHA.

A typical feed mixture for preparing concentrated EPA (EE) by theprocess of the present invention comprises 50-75% EPA (EE), 0 to 10% DHA(EE), and other components including other essential ω-3 and ω-6 fattyacids.

A preferred feed mixture for preparing concentrated EPA (EE) by theprocess of the present invention comprises 55% EPA (EE), 5% DHA (EE),and other components including other essential ω-3 and ω-6 fatty acids.DHA (EE) is less polar than EPA(EE).

A typical feed mixture for preparing concentrated DHA (EE) by theprocess of the present invention comprises 50-75% DHA (EE), 0 to 10% EPA(EE), and other components including other essential ω-3 and ω-6 fattyacids.

A preferred feed mixture for preparing concentrated DHA (EE) by theprocess of the present invention comprises 75% DHA (EE), 7% EPA (EE) andother components including other essential ω-3 and ω-6 fatty acids. EPA(EE) is more polar than DHA (EE).

A typical feed mixture for preparing a concentrated mixture of EPA (EE)and DHA (EE) by the process of the present invention comprises greaterthan 33% EPA (EE), and greater than 22% DHA (EE).

The process of the present invention involves at least twochromatographic separation steps, where a mixture of water and adifferent organic solvent is used as eluent in each step. The first andsecond separation steps are carried out using mixtures of water andfirst and second organic solvents respectively.

Typically, neither eluent is in a supercritical state. Typically, botheluents are liquids.

The first and second organic solvents are typically chosen fromalcohols, ethers, esters, ketones and nitriles. Alcohols and nitrilesare preferred.

Alcohol solvents are well known to the person skilled in the art.Alcohols are typically short chain alcohols. Alcohols typically are offormula ROH, wherein R is a straight or branched C1-C6 alkyl group. TheC1-C6 alkyl group is preferably unsubstituted. Examples of alcoholsinclude methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol,s-butanol and t-butanol. Methanol and ethanol are preferred. Methanol ismore preferred.

Ether solvents are well known to the person skilled in the art. Ethersare typically short chain ethers. Ethers typically are of formulaR—O—R′, wherein R and R′ are the same or different and represent astraight or branched C1-C6 alkyl group. The C1-C6 alkyl group ispreferably unsubstituted. Preferred ethers include diethylether,diisopropylether, and methyl t-butyl ether (MTBE).

Ester solvents are well known to the person skilled in the art. Estersare typically short chain esters. Esters typically are of formulaR—(C═O)O—R′, wherein R and R′ are the same or different and represent astraight or branched C1-C6 alkyl group. Preferred esters includemethylacetate and ethylacetate.

Ketone solvents are well known to the person skilled in the art. Ketonesare typically short chain ketones. Ketones typically are of formulaR—(C═O)—R′, wherein R and R′ are the same or different and represent astraight or branched C1-C6 alkyl group. The C1-C6 alkyl group ispreferably unsubstituted. Preferred ketones include acetone,methylethylketone and methyl isobutyl ketone (MIBK).

Nitrile solvents are well known to the person skilled in the art.Nitriles are typically short chain nitriles. Nitriles typically are offormula R—CN, wherein R represents a straight or branched C1-C6 alkylgroup. The C1-C6 alkyl group is preferably unsubstituted. Preferrednitriles include acetonitrile.

The second organic solvent is different from the first organic solvent.

The polarity index (P′) of a solvent is a well-known measure of howpolar a solvent is. A higher polarity index figure indicates a morepolar solvent. Polarity index is typically determined by measuring theability of a solvent to interact with various test solutes. Moretypically, the polarity index (P′) of a solvent is as defined in Burdickand Jackson's Solvent Guide (AlliedSignal, 1997), the entirety of whichis incorporated herein by reference. Burdick and Jackson rank solventsby reference to a numerical index that ranks solvents according to theirdifferent polarity. The Burdick and Jackson index is based on thestructure of the solvents.

The polarity index (F) of a variety of common solvents is set out in theTable below, which is in accordance with Burdick and Jackson.

Solvent Polarity Index (P′) Pentane 0.0 1,1,2-Trichlorotrifluoroethane0.0 Cyclopentane 0.1 Heptane 0.1 Hexane 0.1 Iso-Octane 0.1 PetroleumEther 0.1 Cyclohexane 0.2 n-Butyl Chloride 1.0 Toluene 2.4 Methylt-Butyl Ether 2.5 o-Xylene 2.5 Chlorobenzene 2.7 o-Dichlorobenzene 2.7Ethyl Ether 2.8 Dichloromethane 3.1 Ethylene Dichloride 3.5 n-ButylAlcohol 3.9 Isopropyl Alcohol 3.9 n-Butyl Acetate 4.0 Isobutyl Alcohol4.0 Methyl Isoamyl Ketone 4.0 n-Propyl Alcohol 4.0 Tetrahydrofuran 4.0Chloroform 4.1 Methyl Isobutyl Ketone 4.2 Ethyl Acetate 4.4 Methyln-Propyl Ketone 4.5 Methyl Ethyl Ketone 4.7 1,4-Dioxane 4.8 Acetone 5.1Methanol 5.1 Ethanol 5.2 Pyridine 5.3 2-Methoxyethanol 5.5 Acetonitrile5.8 Propylene Carbonate 6.1 N,N-Dimethylformamide 6.4 Dimethyl Acetamide6.5 N-Methylpyrrolidone 6.7 Dimethyl Sulfoxide 7.2 Water 10.2

The second organic solvent has a polarity index which differs from thepolarity index of the first organic solvent by between 0.1 and 2.0.Thus, where the polarity index of the first organic solvent is P1, thepolarity index of the second organic solvent is P2, |P1−P2| is 0.1 to2.0.

Typically, the second organic solvent has a polarity index which differsfrom the polarity index of the first organic solvent by at least 0.2,preferably at least 0.3, more preferably at least 0.4, still morepreferably at least 0.5, and yet more preferably at least 0.6.

Typically, the second organic solvent has a polarity index which differsfrom the polarity index of the first organic solvent by at most 1.8,preferably at most 1.5, more preferably at most 1.3, still morepreferably at most 1.0, and yet more preferably at most 0.8.

Preferably, the second organic solvent has a polarity index whichdiffers from the polarity index of the first organic solvent by between0.2 and 1.8, more preferably by between 0.3 and 1.5, still morepreferably by between 0.4 and 1.3, yet more preferably by between 0.5and 1.0, and most preferably by between 0.6 and 0.8.

Typically, the first and second organic solvents are miscible withwater. More typically, the first and second organic solvents have apolarity index of 3.9 or greater. Preferably, the first and secondorganic solvents are chosen from tetrahydrofuran, isopropyl alcohol,n-propyl alcohol, methanol, ethanol, acetonitrile, 1,4-dioxane,N,N-dimethyl formamide, and dim ethyl sulphoxide.

Typically, the first organic solvent:water ratio is from 99.9:0.1 to75:25 parts by volume, preferably from 99.5:0.5 to 80:20 parts byvolume. If the first organic solvent is methanol, the methanol:waterratio is typically from 99.9:0.1 to 85:15 parts by volume, preferablyfrom 99.5:0.5 to 88:12 parts by volume. If the first organic solvent isacetonitrile, the acetonitrile:water ratio is typically from 99:1 to75:25 parts by volume, preferably from 96:4 to 80:20 parts by volume.

Typically, the second organic solvent:water ratio is from 99.9:0.1 to75:25 parts by volume, preferably from 93:7 to 85:15 parts by volume. Ifthe second organic solvent is methanol, the methanol:water ratio istypically from 95:5 to 85:15 parts by volume, preferably from 93:7 to90:10 parts by volume. If the second organic solvent is acetonitrile,the acetonitrile:water ratio is typically from 90:10 to 80:20 parts byvolume, preferably from 88:12 to 85:15 parts by volume.

Typically, one of the first and second organic solvents is acetonitrile.

Typically, one of the first and second organic solvents is methanol.

Preferably, the first and second organic solvents are selected fromacetonitrile and methanol. Thus, it is preferable that (i) the firstorganic solvent is methanol and the second organic solvent isacetonitrile, or (ii) the first organic solvent is acetonitrile and thesecond organic solvent is methanol.

More preferably, the first organic solvent is methanol and the secondorganic solvent is acetonitrile, and (a) the methanol:water ratio isfrom 99.9:0.1 to 85:15 parts by volume, preferably from 99.5:0.5 to88:12 and/or (b) the acetonitrile:water ratio is from 90:10 to 80:20parts by volume, preferably from 88:12 to 85:15 parts by volume. Incertain embodiments it is preferable that (a) the methanol:water ratiois from 91:9 to 93:7 parts by volume, and/or (b) the acetonitrile:waterratio is from 86:14 to 88:12 parts by volume.

Alternatively, the first organic solvent is acetonitrile and the secondorganic solvent is methanol, and (a) the acetonitrile:water ratio isfrom 99:1 to 75:25 parts by volume, preferably 96:4 to 80:20 parts byvolume, and/or (b) the methanol:water ratio is from 95:5 to 85:15 partsby volume, preferably from 93:7 to 90:10 parts by volume. In certainembodiments it is preferable that (a) the acetonitrile:water ratio isfrom 86:14 to 88:12 parts by volume, and/or (b) the methanol:water ratiois from 87:13 to 89:11 parts by volume.

Each chromatographic separation step typically involves passing a feedmixture through one or more chromatographic columns. Thus, the firstchromatographic separation step typically comprises passing the feedmixture through one or more chromatographic columns containing, aseluent, the mixture of water and the first organic solvent. Typically,the second chromatographic separation step comprises passing theintermediate product through one or more chromatographic columnscontaining, as eluent, the mixture of water and the first organicsolvent. Preferably, the first chromatographic separation step comprisespassing the feed mixture through one or more chromatographic columnscontaining, as eluent, the mixture of water and the first organicsolvent, and the second chromatographic separation step comprisespassing the intermediate product through one or more chromatographiccolumns containing, as eluent, the mixture of water and the firstorganic solvent. Any known chromatographic columns may be used in theclaimed process.

The one or more chromatographic columns typically contains an adsorbent.Conventional adsorbents known in the art for chromatographic separationtechniques may be used in the process of the present invention. Whenmore than one chromatographic column is used, each chromatographiccolumn may contain the same or a different adsorbent. Typically, whenmore than one chromatographic column is used each column contains thesame adsorbent. Examples of such commonly used materials are polymericbeads, preferably polystyrene reticulated with DVB (divinylbenzene); andsilica gel, preferably reverse phase bonded silica gel with C8 or C18alkanes, especially C18. C18 bonded reverse phase silica gel ispreferred. The adsorbent used in the process of the present invention ispreferably non-polar.

The shape of the adsorbent stationary phase material may be, forexample, spherical or nonspherical beads, preferably substantiallyspherical beads. Such beads typically have a diameter of 5 to 500microns, preferably 10 to 500 microns, more preferably 15 to 500microns, more preferably 40 to 500 microns, more preferably 100 to 500microns, more preferably 250 to 500 microns, even more preferably 250 to400 microns, most preferably 250 to 350 microns. In some embodiments,beads with a diameter of 5 to 35 microns may be used, typically 10 to 30microns, preferably 15 to 25 microns. Some preferred particle sizes aresomewhat larger than particle sizes of beads used in the past insimulated and actual moving bed processes. Use of larger particlesenables a lower pressure of eluent to be used in the system. This, inturn, has advantages in terms of cost savings, efficiency and lifetimeof the apparatus. It has surprisingly been found that adsorbent beads oflarge particle size may be used in the process of the present invention(with their associated advantages) without any loss in resolution.

The dimensions of the columns used are not particularly limited, andwill depend to some extent on the volume of feed mixture to be purified.A skilled person would easily be able to determine appropriately sizedcolumns to use. The diameter of each column is typically between 10 and1000 mm, preferably between 10 and 500 mm, more preferably between 25and 250 mm, even more preferably between 50 and 100 mm, and mostpreferably between 70 and 80 mm. The length of each column is typicallybetween 10 and 300 cm, preferably between 10 and 200 cm, more preferablybetween 25 and 150 cm, even more preferably between 70 and 110 cm, andmost preferably between 80 and 100 cm.

Any known chromatography apparatus may be used for the purposes of eachseparation step. The number of chromatographic columns used in eachseparation step is not particularly limited.

Typically, the process of the invention is carried out at roomtemperature, or a temperature greater than room temperature. Preferably,the process is carried out at a temperature greater than roomtemperature. The first and second separation steps may be carried out atthe same temperature or a different temperature, preferably the sametemperature.

Typically, the temperature of at least one of the chromatographiccolumns through which the feed mixture is passed is greater than roomtemperature. More typically, the temperature of all of thechromatographic columns used is greater than room temperature.

Thus, typically each chromatographic separation step involves passing afeed mixture through one or more chromatographic columns, and thetemperature of at least one of those chromatographic columns is greaterthan room temperature. More typically, the temperature of all of thechromatographic columns used is greater than room temperature.

As will be appreciated, if at least one chromatographic column is at atemperature greater than room temperature, it is the interior of thecolumn which is important to the separation process. Thus, it istypically the eluent and adsorbent inside the chromatographic columnwhich may be at the temperature greater than room temperature. It is, ofcourse, possible to achieve the required temperature inside the at leastone chromatographic column by internal (for example by heating theeluent and/or feed mixture) and/or external means (for example byheating the outside of the chromatographic column by any knownconventional means).

Typically, an elevated temperature can be achieved by heating the eluentand/or feed mixture. This has the effect of heating the columnsinternally.

Thus, the temperature of at least one of the chromatographic columnsthrough which the feed mixture is passed can also be measured as thetemperature of the eluent. Typically, therefore, the temperature of theeluent used in the first and/or second chromatographic separation stepsis greater than room temperature.

Alternatively, the required temperature of at least one of thechromatographic columns may be achieved by heating the columns. Theheating may be carried out using, for example, an electric heatingmantle, a heated water jacket or coil or by radiative heat lamps. Theinterior and/or exterior of the one or more chromatographic columns maytypically be heated.

The required temperature of at least one of the chromatographic columnsmay be achieved by heating the columns and/or the aqueous organicsolvent eluent, and/or the feed mixture.

Typically, the temperature greater than room temperature is greater than30° C., preferably greater than 35° C., more preferably greater than 40°C., even more preferably greater than 45° C., even more preferablygreater than 50° C., even more preferably greater than 55° C., and evenmore preferably greater than 57° C. A temperature of 56° C. is useful incertain embodiments.

Typically, the temperature greater than room temperature is up to 100°C., preferably up to 95° C., more preferably up to 90° C., even morepreferably up to 85° C., even more preferably up to 80° C., even morepreferably up to 75° C., and even more preferably up to 70° C.

Thus, typical temperature ranges are from 30 to 100° C., from 35 to 95°C., from 40 to 90° C., from 45 to 85° C., from 50 to 80° C., from 55 to75° C. or from 57 to 70° C.

Preferred temperature ranges are from 40 to 70° C., preferably from 50to 67° C., more preferably from 56 to 65° C., even more preferably from57 to 63° C.

In certain embodiments a single chromatographic column may be used,preferably a single stationary chromatographic column. Separation inthis manner is typically carried out using known stationary bedchromatography apparatuses. Separation in this manner may be referred toas “stationary bed” chromatography. Typically, at least one of the firstand/or second chromatographic separation steps involves at least one,for example one, “stationary bed” chromatography step.

In other embodiments, more than one chromatographic column is used. Thismay involve passing the feed mixture through two or more chromatographiccolumns, which may be the same or different, arranged in series or inparallel. The number of columns used in this embodiment is notparticularly limited, but typically does not exceed thirty columns.

One particular embodiment where multiple chromatographic columns areused is simulated or actual moving bed chromatography.

Simulated and actual moving bed chromatography apparatuses are wellknown to the person skilled in the art. Any known simulated or actualmoving bed chromatography apparatus may be utilised for the purposes ofthe method of the present invention, as long as the apparatus is used inaccordance with the process of the present invention. Those apparatusesdescribed in U.S. Pat. No. 2,985,589, U.S. Pat. No. 3,696,107, U.S. Pat.No. 3,706,812, U.S. Pat. No. 3,761,533, FR-A-2103302, FR-A-2651148,FR-A-2651149, U.S. Pat. No. 6,979,402, U.S. Pat. No. 5,069,883 and U.S.Pat. No. 4,764,276 may all be used if configured in accordance with theprocess of the present invention. SMB processes as disclosed in, forexample, WO-A-2011/080503 may also be employed.

The first and second separation steps may be carried out using either astationary bed chromatography apparatus, or one or more simulated oractual moving bed chromatography apparatuses as discussed herein.

Typically, the first chromatographic separation step comprisesintroducing the feed mixture into a stationary bed chromatographyapparatus and the second chromatographic separation step comprisesintroducing the intermediate product into a stationary bedchromatography apparatus. Thus, typically the first chromatographicseparation step is carried out using a stationary bed chromatographyapparatus and the second chromatographic separation step is carried outusing a stationary bed chromatography apparatus.

Alternatively, the first chromatographic separation step comprisesintroducing the feed mixture into a stationary bed apparatus and thesecond chromatographic separation step comprises introducing theintermediate product into a simulated or actual moving bedchromatography apparatus. Thus, typically the first chromatographicseparation step is carried out using a stationary bed apparatus and thesecond chromatographic separation step is carried out using a simulatedor actual moving bed chromatography apparatus.

Alternatively, the first chromatographic separation step comprisesintroducing the feed mixture into a simulated or actual moving bedchromatography apparatus and the second chromatographic separation stepcomprises introducing the intermediate product into a stationary bedchromatography apparatus. Thus, typically the first chromatographicseparation step is carried out using a simulated or actual moving bedchromatography apparatus and the second chromatographic separation stepis carried out using a stationary bed chromatography apparatus.

Alternatively, the first chromatographic separation step comprisesintroducing the feed mixture into a simulated or actual moving bedchromatography apparatus and the second chromatographic separation stepcomprises introducing the intermediate product into a simulated oractual moving bed chromatography apparatus. Thus, typically the firstchromatographic separation step is carried out using a simulated oractual moving bed chromatography apparatus and the secondchromatographic separation step is carried out using a simulated oractual moving bed chromatography apparatus.

Said first chromatographic separation step may consist of a singlechromatographic separation or two or more chromatographic separations,provided that each separation uses as eluent a mixture of water and thefirst organic solvent.

Said second chromatographic separation step may consist of a singlechromatographic separation or two or more chromatographic separations,provided that each separation uses as eluent a mixture of water and thesecond organic solvent.

Typically, the first and/or second chromatographic separation steps caninvolve the use of a single SMB separation step using conventionalapparatus, such as for example depicted in FIG. 1. Separation in thismanner may be referred to as “single pass” SMB. Typically, at least oneof the first and/or second chromatographic separation steps involves atleast one, for example one, “single pass” SMB step.

Alternatively, the first and/or second chromatographic separation stepscan each involve the use of multiple SMB separations.

In one embodiment, the first chromatographic separation step and/or thesecond chromatographic separation step can be carried out as describedin WO-A-2011/080503 and PCT/GB2012/051591, the entirety of which areincorporated herein by reference. Preferred process conditions specifiedin WO-A-2011/080503 and PCT/GB2012/051591 are preferred processconditions for this embodiment, and may be incorporated fromWO-A-2011/080503 and PCT/GB2012/051591.

The process disclosed in WO-A-2011/080503 and PCT/GB2012/051591 involvesintroducing an input stream to a simulated or actual moving bedchromatography apparatus having a plurality of linked chromatographycolumns containing, as eluent, an aqueous organic solvent, wherein theapparatus has a plurality of zones comprising at least a first zone andsecond zone, each zone having an extract stream and a raffinate streamfrom which liquid can be collected from said plurality of linkedchromatography columns, and wherein (a) a raffinate stream containingthe PUFA product together with more polar components is collected from acolumn in the first zone and introduced to a nonadjacent column in thesecond zone, and/or (b) an extract stream containing the PUFA producttogether with less polar components is collected from a column in thesecond zone and introduced to a nonadjacent column in the first zone,said PUFA product being separated from different components of the inputstream in each zone. Separation in this manner may be referred to as a“double pass” SMB process.

In this “double pass” SMB process, the term “zone” refers to a pluralityof linked chromatography columns containing, as eluent, an aqueousorganic solvent, and having one or more injection points for an inputstream, one or more injection points for water and/or organic solvent, araffinate take-off stream from which liquid can be collected from saidplurality of linked chromatography columns, and an extract take-offstream from which liquid can be collected from said plurality of linkedchromatography columns. Typically, each zone has only one injectionpoint for an input stream. In one embodiment, each zone has only oneinjection point for the aqueous organic solvent eluent. In anotherembodiment, each zone has two or more injection points for water and/ororganic solvent.

In this “double pass” SMB process, reference to an “input stream” refersto the feed mixture when the above-described SMB process is used in thefirst chromatographic separation step, and refers to the intermediateproduct when the above-described SMB process is used in the secondchromatographic separation step.

In this “double pass” SMB process, reference to an “aqueous organicsolvent” refers to the mixture of water and the first organic solventwhen the above-described SMB process is used in the firstchromatographic separation step, and refers to the mixture of water andthe second organic solvent when the above-described SMB process is usedin the second chromatographic separation step.

The term “raffinate” is well known to the person skilled in the art. Inthe context of actual and simulated moving bed chromatography it refersto the stream of components that move more rapidly with the liquideluent phase compared with the solid adsorbent phase. Thus, a raffinatestream is typically enriched with more polar components, and depleted ofless polar components compared with an input stream.

The term “extract” is well known to the person skilled in the art. Inthe context of actual and simulated moving bed chromatography it refersto the stream of components that move more rapidly with the solidadsorbent phase compared with the liquid eluent phase. Thus, an extractstream is typically enriched with less polar components, and depleted ofmore polar components compared with an input stream.

As used herein, the term “nonadjacent” refers to columns, in for examplethe same apparatus, separated by one or more columns, preferably 3 ormore columns, more preferably 5 or more columns, most preferably about 5columns.

The “double pass” SMB process is illustrated in FIG. 11. An input streamF comprising the PUFA product (B) and more polar (C) and less polar (A)components is introduced into the top of column 5 in the first zone.Aqueous organic solvent desorbent is introduced into the top of column 1in the first zone. In the first zone, the less polar components (A) areremoved as extract stream E1 from the bottom of column 2. The PUFAproduct (B) and more polar components (C) are removed as raffinatestream R1 from the bottom of column 7. Raffinate stream R1 is thenintroduced into the second zone at the top of column 12. Aqueous organicsolvent desorbent is introduced into the top of column 9 in the secondzone. In the second zone, the more polar components (C) are removed asraffinate stream R2 at the bottom of column 14. The PUFA product (B) iscollected as extract stream E2 at the bottom of column 10.

In this “double pass” SMB process, aqueous organic solvent is typicallyintroduced into the top of column 1 in the first zone.

In this “double pass” SMB process, aqueous organic solvent is typicallyintroduced into the top of column 9 in the second zone.

In this “double pass” SMB process, the input stream is typicallyintroduced into the top of column 5 in the first zone.

In this “double pass” SMB process, a first raffinate stream is typicallycollected from the bottom of column 7 in the first zone and introducedinto the top of column 12 in the second zone. The first raffinate streammay optionally be collected in a container before being introduced intocolumn 12.

In this “double pass” SMB process, a first extract stream is typicallyremoved from the bottom of column 2 in the first zone. The first extractstream may optionally be collected in a container and a portionreintroduced into the top of column 3 in the first zone. The rate ofrecycle of liquid collected via the extract stream from the first zoneback into the first zone is the rate at which liquid is pumped from thiscontainer into the top of column 3.

In this “double pass” SMB process, a second raffinate stream istypically removed from the bottom of column 14 in the second zone.

In this “double pass” SMB process, a second extract stream is typicallycollected from the bottom of column 10 in the second zone. This secondextract stream typically contains the PUFA product. The second extractstream may optionally be collected in a container and a portionreintroduced into the top of column 11 in the second zone. The rate ofrecycle of liquid collected via the extract stream from the second zoneback into the second zone is the rate at which liquid is pumped fromthis container into the top of column 11.

In this “double pass” SMB process, the rate at which liquid collectedvia the extract stream from the first zone is recycled back into thefirst zone is typically faster than the rate at which liquid collectedvia the extract stream from the second zone is recycled back into thesecond zone. In this “double pass” SMB process, eluent is typicallysubstantially the same in each zone.

Typically, at least one of the first and second chromatographicseparation steps involves at least one, for example one, “double pass”SMB process as defined above.

In an alternative embodiment, the first chromatographic separation stepand/or the second chromatographic separation step can be carried out asdescribed in international patent application no. PCT/GB2012/051596 orPCT/GB2012/051597, the entirety of which are incorporated herein byreference. Such embodiments involve (i) purifying an input stream in afirst SMB step in a simulated or actual moving bed chromatographyapparatus having a plurality of linked chromatography columnscontaining, as eluent, an aqueous organic solvent, to obtain a firstproduct; and

(ii) purifying the first product obtained in (i) in a second SMB stepusing a simulated or actual moving bed chromatography apparatus having aplurality of linked chromatography columns containing, as eluent, anaqueous organic solvent, to obtain a second product; wherein

(a) the first and second SMB steps are carried out sequentially on thesame chromatography apparatus, the first product being recovered betweenthe first and second SMB steps and the process conditions in thechromatography apparatus being adjusted between the first and second SMBsteps such that the PUFA product is separated from different componentsof the feed mixture in each SMB step; or

(b) the first and second SMB steps are carried out on separate first andsecond chromatography apparatuses respectively, the first productobtained from the first SMB step being introduced into the secondchromatography apparatus, and the PUFA product being separated fromdifferent components of the feed mixture in each SMB step. Separation inthis manner by be referred to as “back-to-back” SMB.

For the avoidance of doubt, if the first chromatographic separation stepis a “back-to-back” SMB process along the above lines, the eluent ineach of the SMB steps is a mixture of water and the first organicsolvent. If the second chromatographic separation step is a“back-to-back” SMB process along the above lines, the eluent in each ofthe SMB steps is a mixture of water and the second organic solvent.

In this “back-to-back” SMB process, the term “simulated or actual movingbed chromatography apparatus” typically refers to a plurality of linkedchromatography columns containing, as eluent, an aqueous organicsolvent, and having one or more injection points for an input stream,one or more injection points for water and/or organic solvent, araffinate take-off stream from which liquid can be collected from saidplurality of linked chromatography columns, and an extract take-offstream from which liquid can be collected from said plurality of linkedchromatography columns.

The chromatography apparatus used in this “back-to-back” SMB process hasa single array of chromatography columns linked in series containing, aseluent, an aqueous organic solvent. Typically, each of thechromatography columns are linked to the two columns in the apparatusadjacent to that column. Thus, the output from a given column in thearray is connected to the input of the adjacent column in the array,which is downstream with respect to the flow of eluent in the array.Thus, eluent can flow around the array of linked chromatography columns.Typically, none of the chromatography columns are linked to non-adjacentcolumns in the apparatus.

In this “back-to-back” SMB process, reference to an “input stream”refers to the feed mixture when the above-described SMB process is usedin the first chromatographic separation step, and refers to theintermediate product when the above-described SMB process is used in thesecond chromatographic separation step.

In this “back-to-back” SMB process, reference to an “aqueous organicsolvent” refers to the mixture of water and the first organic solventwhen the above-described “back-to-back” SMB process is used in the firstchromatographic separation step, and refers to the mixture of water andthe second organic solvent when the above-described “back-to-back” SMBprocess is used in the second chromatographic separation step. Theorganic solvent used in the first and second SMB steps is the same. Theorganic solvent:water ratio used in the first and second SMB steps maybe the same or different.

In this “back-to-back” SMB process, reference to a “second product”refers to the intermediate product when the above-described SMB processis used in the first chromatographic separation step, and refers to thePUFA product when the above-described SMB process is used in the secondchromatographic separation step.

Typically in this “back-to-back” SMB process, each apparatus has onlyone injection point for an input stream. In one embodiment, eachapparatus has only one injection point for the aqueous organic solventeluent. In another embodiment, each apparatus has two or more injectionpoints for water and/or organic solvent.

The term “raffinate” is well known to the person skilled in the art. Inthe context of actual and simulated moving bed chromatography it refersto the stream of components that move more rapidly with the liquideluent phase compared with the solid adsorbent phase. Thus, a raffinatestream is typically enriched with more polar components, and depleted ofless polar components compared with a feed stream.

The term “extract” is well known to the person skilled in the art. Inthe context of actual and simulated moving bed chromatography it refersto the stream of components that move more rapidly with the solidadsorbent phase compared with the liquid eluent phase. Thus, an extractstream is typically enriched with less polar components, and depleted ofmore polar components compared with a feed stream.

The number of columns used in each apparatus in this “back-to-back” SMBprocess is not particularly limited. A skilled person would easily beable to determine an appropriate number of columns to use. The number ofcolumns is typically 4 or more, preferably 6 or more, more preferably 8or more, for example 4, 5, 6, 7, 8, 9, or 10 columns. In a preferredembodiment, 5 or 6 columns, more preferably 6 columns are used. Inanother preferred embodiment, 7 or 8 columns, more preferably 8 columnsare used. Typically, there are no more than 25 columns, preferably nomore than 20, more preferably no more than 15.

In this “back-to-back” SMB process, the chromatographic apparatuses usedin the first and second separation steps typically contain the samenumber of columns. For certain applications they may have differentnumbers of columns.

In this “back-to-back” SMB process, the columns in the chromatographicapparatuses used in the first and second SMB separation steps typicallyhave identical dimensions but may, for certain applications, havedifferent dimensions.

The flow rates to the columns are limited by maximum pressures acrossthe series of columns and will depend on the column dimensions andparticle size of the solid phases. One skilled in the art will easily beable to establish the required flow rate for each column dimension toensure efficient desorption. Larger diameter columns will in generalneed higher flows to maintain linear flow through the columns.

In this “back-to-back” SMB process, for the typical column sizesoutlined above, typically the flow rate of eluent into thechromatographic apparatus used in the first SMB separation step is from1 to 4.5 L/min, preferably from 1.5 to 2.5 L/min. Typically, the flowrate of the extract from the chromatographic apparatus used in the firstSMB separation step is from 0.1 to 2.5 L/min, preferably from 0.5 to2.25 L/min. In embodiments where part of the extract from the first SMBseparation step is recycled back into the apparatus used in the firstSMB separation step, the flow rate of recycle is typically from 0.7 to1.4 L/min, preferably about 1 L/min. Typically, the flow rate of theraffinate from the chromatographic apparatus used in the first SMBseparation step is from 0.2 to 2.5 L/min, preferably from 0.3 to 2.0L/min. In embodiments where part of the raffinate from the first SMBseparation step is recycled back into the apparatus used in the firstSMB separation step, the flow rate of recycle is typically from 0.3 to1.0 L/min, preferably about 0.5 L/min. Typically, the flow rate ofintroduction of the input stream into the chromatographic apparatus usedin the first SMB separation step is from 5 to 150 mL/min, preferablyfrom 10 to 100 mL/min, more preferably from 20 to 60 mL/min.

In this “back-to-back” SMB process, for the typical column sizesoutlined above, typically the flow rate of eluent into thechromatographic apparatus used in the second SMB separation step is from1 to 4 L/min, preferably from 1.5 to 3.5 L/min. Typically, the flow rateof the extract from the chromatographic apparatus used in the second SMBseparation step is from 0.5 to 2 L/min, preferably from 0.7 to 1.9L/min. In embodiments where part of the extract from the second SMBseparation step is recycled back into the apparatus used in the secondSMB separation step, the flow rate of recycle is typically from 0.6 to1.4 L/min, preferably from 0.7 to 1.1 L/min, more preferably about 0.9L/min. Typically, the flow rate of the raffinate from thechromatographic apparatus used in the second SMB separation step is from0.5 to 2.5 L/min, preferably from 0.7 to 1.8 L/min, more preferablyabout 1.4 L/min. In embodiments where part of the raffinate from thesecond SMB separation step is recycled back into the apparatus used inthe second SMB separation step, the flow rate of recycle is typicallyfrom 0.3 to 1.0 L/min, preferably about 0.5 L/min.

As the skilled person will appreciate, references to rates at whichliquid is collected or removed via the various extract and raffinatestreams refer to volumes of liquid removed in an amount of time,typically L/minute. Similarly, references to rates at which liquid isrecycled back into an apparatus, typically to an adjacent column in theapparatus, refer to volumes of liquid recycled in an amount of time,typically L/minute.

In this “back-to-back” SMB process, actual moving bed chromatography ispreferred.

The step time, i.e. the time between shifting the points of injection ofthe input stream and eluent, and the various take off points of thecollected fractions, is not particularly limited, and will depend on thenumber and dimensions of the columns used, and the flow rate through theapparatus. A skilled person would easily be able to determineappropriate step times to use in the process of the present invention.The step time is typically from 100 to 1000 seconds, preferably from 200to 800 seconds, more preferably from about 250 to about 750 seconds. Insome embodiments, a step time of from 100 to 400 seconds, preferably 200to 300 seconds, more preferably about 250 seconds, is appropriate. Inother embodiments, a step time of from 600 to 900 seconds, preferably700 to 800 seconds, more preferably about 750 seconds is appropriate.

The “back-to-back” SMB process comprises a first and second SMBseparation step.

These two steps can easily be carried out on a single chromatographicapparatus. Thus, in one embodiment, (a) the first and second SMBseparation steps are carried out sequentially on the same chromatographyapparatus, the first product being recovered between the first andsecond SMB separation steps and the process conditions in thechromatography apparatus being adjusted between the first and second SMBseparation steps such that the PUFA product is separated from differentcomponents of the input stream in each separation step. A preferredembodiment of this “back-to-back” SMB process is shown as FIG. 10a .Thus, the first SMB separation step (left hand side) is carried out onan SMB apparatus having 8 columns. Between the first and second SMBseparation steps the first product is recovered in, for example, acontainer, the process conditions in the chromatography apparatus areadjusted such that the PUFA product is separated from differentcomponents of the input stream in each SMB separation step. The secondSMB separation step (right hand side) is then carried out on the sameSMB apparatus having 8 columns.

In embodiment (a), adjusting the process conditions typically refers toadjusting the process conditions in the apparatus as a whole, i.e.physically modifying the apparatus so that the conditions are different.It does not refer to simply reintroducing the first product back into adifferent part of the same apparatus where the process conditions mighthappen to be different.

Alternatively, first and second separate chromatographic apparatuses canbe used in the first and second SMB separation steps. Thus, in anotherembodiment, (b) the first and second SMB separation steps are carriedout on separate first and second chromatography apparatusesrespectively, the first product obtained from the first SMB separationstep being introduced into the second chromatography apparatus, and thePUFA product being separated from different components of the inputstream in each SMB separation step.

In embodiment (b), the two SMB separation steps may either be carriedout sequentially or simultaneously.

Thus, in embodiment (b) in the case where the two SMB separation stepsare carried out sequentially, the first and second SMB separation stepsare carried out sequentially on separate first and second chromatographyapparatuses respectively, the first product being recovered between thefirst and second SMB separation steps and the process conditions in thefirst and second SMB chromatography apparatuses being adjusted such thatthe PUFA product is separated from different components of the inputstream in each separation step. A preferred embodiment of this“back-to-back” SMB separation process is shown as FIG. 10b . Thus, thefirst SMB separation step (left hand side) is carried out on an SMBapparatus having 8 columns, one to eight. Between the first and secondSMB separation steps the first product is recovered, for example in acontainer, and then introduced into a second separate SMB apparatus. Thesecond SMB separation step (right hand side) is carried out on thesecond separate SMB apparatus which has 8 columns, nine to sixteen. Theprocess conditions in the two chromatography apparatuses are adjustedsuch that the PUFA product is separated from different components of theinput stream in each SMB separation step.

In embodiment (b) in the case where the two SMB separation steps arecarried our simultaneously, the first and second SMB separation stepsare carried out on separate first and second chromatography apparatusesrespectively, the first product being introduced into the chromatographyapparatus used in the second SMB separation step, and the processconditions in the first and second chromatography apparatuses beingadjusted such that the PUFA product is separated from differentcomponents of the input stream in each SMB separation step. A preferredembodiment of this “back-to-back” SMB separation process is shown asFIG. 10c . Thus, the first SMB separation step (left hand side) iscarried out on an SMB apparatus having 8 columns, one to eight. Thefirst product obtained in the first SMB separation step is thenintroduced into the second separate chromatography apparatus used in thesecond SMB separation step. The first product may be passed from thefirst SMB separation step to the second SMB separation step directly orindirectly, for example via a container. The second SMB separation step(right hand side) is carried out on the second separate SMB apparatuswhich has 8 columns, nine to sixteen. The process conditions in the twochromatography apparatuses are adjusted such that the PUFA product isseparated from different components of the input stream in eachseparation step.

In embodiment (b) in the case where the two SMB separation steps arecarried our simultaneously, eluent circulates separately in the twoseparate chromatographic apparatuses. Thus, eluent is not shared betweenthe two separate chromatographic apparatuses other than what eluent maybe present as solvent in the first product which is purified in thesecond SMB separation step, and which is introduced into thechromatographic apparatus used in the second SMB separation step.Chromatographic columns are not shared between the two separatechromatographic apparatuses used in the first and second SMB separationsteps.

In this “back-to-back” SMB process, after the first product is obtainedin the first SMB separation step, the aqueous organic solvent eluent maybe partly or totally removed before the first product is purified in thesecond SMB separation step. Alternatively, the first product may bepurified in the second SMB separation step without the removal of anysolvent present.

As mentioned above, in this “back-to-back” SMB process the PUFA productis separated from different components of the input stream in each SMBseparation step. In embodiment (a), the process conditions of the singleSMB apparatus used in both SMB separation steps are adjusted between thefirst and second SMB separation steps such that the PUFA product isseparated from different components of the input stream in eachseparation step. In embodiment (b), the process conditions in the twoseparate chromatography apparatuses used in the first and second SMBseparation steps are set such that the PUFA product is separated fromdifferent components of the input stream in each separation step.

Thus, in this “back-to-back” SMB process the process conditions in thefirst and second SMB separation steps vary. The process conditions whichvary may include, for example, the size of the columns used, the numberof columns used, the packing used in the columns, the step time of theSMB apparatus, the temperature of the apparatus, the water:organicsolvent ration of the eluent used in the separation steps, or the flowrates used in the apparatus, in particular the recycle rate of liquidcollected via the extract or raffinate streams.

Preferably in this “back-to-back” SMB process, the process conditionswhich may vary are the water:organic solvent ratio of the eluent used inthe SMB separation steps, and/or the recycle rate of liquid collectedvia the extract or raffinate streams in the SMB separation steps. Bothof these options are discussed in more detail below.

In this “back-to-back” SMB process, the first product obtained in thefirst SMB separation step is typically enriched in the PUFA productcompared to the input stream.

In this “back-to-back” SMB process, the first product obtained in thefirst SMB separation step is then introduced into the chromatographicapparatus used in the second SMB separation step.

In this “back-to-back” SMB process, the first product is typicallycollected as the raffinate or extract stream from the chromatographicapparatus used in the first SMB separation process.

Typically in this “back-to-back” SMB process, the first product iscollected as the raffinate stream in the first SMB separation step, andthe second product is collected as the extract stream in the second SMBseparation step. Thus, the raffinate stream collected in the first SMBseparation step is used as the input stream in the second SMB separationstep. The raffinate stream collected in the first SMB separation steptypically contains the second product together with more polarcomponents.

Alternatively in this “back-to-back” SMB process, the first product iscollected as the extract stream in the first SMB separation step, andthe second product is collected as the raffinate stream in the secondSMB separation step. Thus, the extract stream collected in the first SMBseparation step is used as the input stream in the second SMB separationstep. The extract stream collected in the first SMB separation steptypically contains the second product together with less polarcomponents.

In this “back-to-back” SMB process the PUFA product is separated fromdifferent components of the input stream in each SMB separation step.Typically, the components separated in each SMB separation step of theprocess of the present invention have different polarities.

Preferably in this “back-to-back” SMB process, the PUFA product isseparated from less polar components of the input stream in the firstSMB separation step, and the PUFA product is separated from more polarcomponents of the input stream in the second SMB separation step.

Typically in this “back-to-back” SMB process, (a) part of the extractstream from the apparatus used in the first SMB separation step isrecycled back into the apparatus used in the first SMB separation step;and/or (b) part of the raffinate stream from the apparatus used in thefirst SMB separation step is recycled back into the apparatus used inthe first SMB separation step; and/or (c) part of the extract streamfrom the apparatus used in the second SMB separation step is recycledback into the apparatus used in the second SMB separation step; and/or(d) part of the raffinate stream from the apparatus used in the secondSMB separation step is recycled back into the apparatus used in thesecond SMB separation step.

Preferably in this “back-to-back” SMB process, (a) part of the extractstream from the apparatus used in the first SMB separation step isrecycled back into the apparatus used in the first SMB separation step;and (b) part of the raffinate stream from the apparatus used in thefirst SMB separation step is recycled back into the apparatus used inthe first SMB separation step; and (c) part of the extract stream fromthe apparatus used in the second SMB separation step is recycled backinto the apparatus used in the second SMB separation step; and (d) partof the raffinate stream from the apparatus used in the second SMBseparation step is recycled back into the apparatus used in the secondSMB separation step.

The recycle in this “back-to-back” SMB process involves feeding part ofthe extract or raffinate stream out of the chromatography apparatus usedin the first or second SMB separation step back into the apparatus usedin that SMB step, typically into an adjacent column. This adjacentcolumn is the adjacent column which is downstream with respect to theflow of eluent in the system.

In this “back-to-back” SMB process the rate at which liquid collectedvia the extract or raffinate stream in the first or second SMBseparation steps is recycled back into the chromatography apparatus usedin that SMB step is the rate at which liquid collected via that streamis fed back into the apparatus used in that SMB step, typically into anadjacent column, i.e. the downstream column with respect to the flow ofeluent in the system.

This can be seen with reference to FIG. 9. The rate of recycle ofextract in the first SMB separation step is the rate at which extractcollected from the bottom of column 2 of the chromatographic apparatusused in the first SMB separation step is fed into the top of column 3 ofthe chromatographic apparatus used in the first SMB separation step,i.e. the flow rate of liquid into the top of column 3 of thechromatographic apparatus used in the first SMB separation step.

In this “back-to-back” SMB process the rate of recycle of extract in thesecond SMB separation step is the rate at which extract collected at thebottom of column 2 of the chromatographic apparatus used in the secondSMB separation step is fed into the top of column 3 of thechromatographic apparatus used in the second SMB separation step, i.e.the flow rate of liquid into the top of column 3 of the chromatographicapparatus used in the second SMB separation step.

In this “back-to-back” SMB process recycle of the extract and/orraffinate streams in the first and/or second SMB separation steps istypically effected by feeding the liquid collected via that stream inthat SMB separation step into a container, and then pumping an amount ofthat liquid from the container back into the apparatus used in that SMBseparation step, typically into an adjacent column. In this case, therate of recycle of liquid collected via a particular extract orraffinate stream in the first and/or second SMB separation steps,typically back into an adjacent column, is the rate at which liquid ispumped out of the container back into the chromatography apparatus,typically into an adjacent column.

As the skilled person will appreciate, in this “back-to-back” SMBprocess the amount of liquid being introduced into a chromatographyapparatus via the eluent and input streams is balanced with the amountof liquid removed from the apparatus, and recycled back into theapparatus.

Thus, in this “back-to-back” SMB process with reference to FIG. 9, forthe extract stream, the flow rate of eluent (desorbent) into thechromatographic apparatus(es) used in the first and second SMBseparation steps (D) is equal to the rate at which liquid collected viathe extract stream in that SMB separation step accumulates in acontainer (E1 and E2) added to the rate at which extract is recycledback into the chromatographic apparatus used in that particular SMBseparation step (D−E1 and D−E2).

In this “back-to-back” SMB process, for the raffinate stream from a SMBseparation step, the rate at which extract is recycled back into thechromatographic apparatus used in that particular SMB separation step(D−E1 and D−E2) added to the rate at which feedstock is introduced intothe chromatographic apparatus used in that particular SMB separationstep (F and R1) is equal to the rate at which liquid collected via theraffinate stream in that particular SMB separation step accumulates in acontainer (R1 and R2) added to the rate at which raffinate is recycledback into the chromatographic apparatus used in that particular SMBseparation step (D+F−E1−R1 and D+R1−E2−R2).

In this “back-to-back” SMB process, the rate at which liquid collectedfrom a particular extract or raffinate stream from a chromatographyapparatus accumulates in a container can also be thought of as the netrate of removal of that extract or raffinate stream from thatchromatography apparatus.

Typically in this “back-to-back” SMB process, the rate at which liquidcollected via the extract and raffinate streams in the first SMBseparation step is recycled back into the apparatus used in thatseparation step is adjusted such that the PUFA product can be separatedfrom different components of the input stream in each SMB separationstep.

Typically in this “back-to-back” SMB process, the rate at which liquidcollected via the extract and raffinate streams in the second SMBseparation step is recycled back into the apparatus used in that SMBseparation step is adjusted such that the PUFA product can be separatedfrom different components of the input stream in each SMB separationstep.

Preferably in this “back-to-back” SMB process, the rate at which liquidcollected via the extract and raffinate streams in each SMB separationstep is recycled back into the apparatus used in that SMB separationstep is adjusted such that the PUFA product can be separated fromdifferent components of the input stream in each SMB separation step.

Typically in this “back-to-back” SMB process, the rate at which liquidcollected via the extract stream in the first SMB separation step isrecycled back into the chromatography apparatus used in the first SMBseparation step differs from the rate at which liquid collected via theextract stream in the second SMB separation step is recycled back intothe chromatography apparatus used in the second SMB separation step,and/or the rate at which liquid collected via the raffinate stream inthe first SMB separation step is recycled back into the chromatographyapparatus used in the first SMB separation step differs from the rate atwhich liquid collected via the raffinate stream in the second SMBseparation step is recycled back into the chromatography apparatus usedin the second SMB separation step.

Varying the rate at which liquid collected via the extract and/orraffinate streams in the first or second SMB separation steps isrecycled back into the apparatus used in that particular SMB separationstep has the effect of varying the amount of more polar and less polarcomponents present in the extract and raffinate streams. Thus, forexample, a lower extract recycle rate results in fewer of the less polarcomponents in that SMB separation step being carried through to theraffinate stream. A higher extract recycle rate results in more of theless polar components in that SMB separation step being carried throughto the raffinate stream.

This can be seen, for example, in FIG. 6. The rate at which liquidcollected via the extract stream in the first SMB separation step isrecycled back into the chromatographic apparatus used in that SMBseparation step (D−E1) will affect to what extent any of component A iscarried through to the raffinate stream in the first SMB separation step(R1).

Typically in this “back-to-back” SMB process, the rate at which liquidcollected via the extract stream in the first SMB separation step isrecycled back into the chromatographic apparatus used in the first SMBseparation step is faster than the rate at which liquid collected viathe extract stream in the second SMB separation step is recycled backinto the chromatographic apparatus used in the second SMB separationstep. Preferably, a raffinate stream containing the second producttogether with more polar components is collected from the first SMBseparation step and purified in a second SMB separation step, and therate at which liquid collected via the extract stream in the first SMBseparation step is recycled back into the chromatographic apparatus usedin the first SMB separation step is faster than the rate at which liquidcollected via the extract stream in the second SMB separation step isrecycled back into the chromatographic apparatus used in the second SMBseparation step.

Alternatively in this “back-to-back” SMB process, the rate at whichliquid collected via the extract stream in the first SMB separation stepis recycled back into the chromatographic apparatus used in the firstSMB separation step is slower than the rate at which liquid collectedvia the extract stream in the second SMB separation step is recycledback into the chromatographic apparatus used in the second SMBseparation step.

Typically in this “back-to-back” SMB process, the rate at which liquidcollected via the raffinate stream in the first SMB separation step isrecycled back into the chromatographic apparatus used in the firstseparation step is faster than the rate at which liquid collected viathe raffinate stream in the second SMB separation step is recycled backinto the chromatographic apparatus used in the second SMB separationstep. Preferably, an extract stream containing the second producttogether with less polar components is collected from the first SMBseparation step and purified in a second SMB separation step, and therate at which liquid collected via the raffinate stream in the first SMBseparation step is recycled back into the chromatographic apparatus usedin the first SMB separation step is faster than the rate at which liquidcollected via the raffinate stream in the second SMB separation step isrecycled back into the chromatographic apparatus used in the second SMBseparation step.

Alternatively in this “back-to-back” SMB process, the rate at whichliquid collected via the raffinate stream in the first SMB separationstep is recycled back into the chromatographic apparatus used in thefirst SMB separation step is slower than the rate at which liquidcollected via the raffinate stream in the second SMB separation step isrecycled back into the chromatographic apparatus used in the second SMBseparation step.

In this “back-to-back” SMB process, where recycle rates are adjustedsuch that the PUFA product can be separated from different components ofthe input stream in each SMB separation step, the water:organic solventratio of the eluents used in each SMB separation step may be the same ordifferent. Typical water:organic solvent ratios of the eluent in eachSMB separation step are as defined above.

Typically in this “back-to-back” SMB process, the aqueous organicsolvent eluent used in each SMB separation step has a differentwater:organic solvent ratio. The organic solvent used in each SMBseparation step is the same. The water:organic solvent ratio used ineach SMB separation step is preferably adjusted such that the PUFAproduct can be separated from different components of the input streamin each SMB separation step.

In this “back-to-back” SMB process, the eluting power of the eluent usedin each of the SMB separation steps is typically different. Preferably,the eluting power of the eluent used in the first SMB separation step isgreater than that of the eluent used in the second SMB separation step.In practice this is achieved by varying the relative amounts of waterand organic solvent used in each SMB separation step.

Depending on the choice of organic solvent, they may be more powerfuldesorbers than water. Alternatively, they may be less powerful desorbersthan water. Acetonitrile and alcohols, for example, are more powerfuldesorbers than water. Thus, when the aqueous organic solvent is aqueousalcohol or acetonitrile, the amount of alcohol or acetonitrile in theeluent used in the first SMB separation step is typically greater thanthe amount of alcohol or acetonitrile in the eluent used in the secondSMB separation step.

Typically in this “back-to-back” SMB process, the water:organic solventratio of the eluent in the first SMB separation step is lower than thewater:organic solvent ratio of the eluent in the second SMB separationstep. Thus, the eluent in the first SMB separation step typicallycontains more organic solvent than the eluent in the second SMBseparation step.

It will be appreciated that the ratios of water and organic solvent ineach SMB separation step referred to above are average ratios within thetotality of the chromatographic apparatus.

Typically in this “back-to-back” SMB process, the water:organic solventratio of the eluent in each SMB separation step is controlled byintroducing water and/or organic solvent into one or more columns in thechromatographic apparatuses used in the SMB separation steps. Thus, forexample, to achieve a lower water:organic solvent ratio in the first SMBseparations step than in the second SMB separation step, water istypically introduced more slowly into the chromatographic apparatus usedin the first SMB separation step than in the second SMB separation step.

Typically in this “back-to-back” SMB process, essentially pure organicsolvent and essentially pure water may be introduced at different pointsin the chromatographic apparatus used in each SMB separation step. Therelative flow rates of these two streams will determine the overallsolvent profile in the chromatographic apparatus. Alternatively in this“back-to-back” SMB process, different mixtures of the organic solventand water may be introduced at different points in each chromatographicapparatus used in each SMB separation step. That will involveintroducing two or more different mixtures of the organic solvent andwater into the chromatographic apparatus used in a particular SMBseparation step, each organic solvent/water mixture having a differentorganic solvent:water ratio. The relative flow rates and relativeconcentrations of the organic solvent/water mixtures in this“back-to-back” SMB process will determine the overall solvent profile inthe chromatographic apparatus used in that SMB separation step.

Preferably in this “back-to-back” SMB process, either (1) the firstproduct containing the second product together with more polarcomponents is collected as the raffinate stream in the first SMBseparation step, and the second product is collected as the extractstream in the second SMB separation step; or (2) the first productcontaining the second product together with less polar components iscollected as the extract stream in the first SMB separation step, andthe second product is collected as the raffinate stream in the secondSMB separation step.

Option (1) is suitable for purifying EPA from an input stream.

Option (1) is illustrated in FIG. 2. An input stream F comprising thesecond product (B) and more polar (C) and less polar (A) components ispurified in the first SMB separation step. In the first SMB separationstep, the less polar components (A) are removed as extract stream E1.The second product (B) and more polar components (C) are collected asraffinate stream R1. Raffinate stream R1 is the first product which isthen purified in the second SMB separation step. In the second SMBseparation step, the more polar components (C) are removed as raffinatestream R2. The second product (B) is collected as extract stream E2.

Option (1) is illustrated in more detail in FIG. 4. FIG. 4 is identicalto FIG. 2, except that the points of introduction of the organic solventdesorbent (D) and water (W) into each chromatographic apparatus areshown. The organic solvent desorbent (D) and water (W) together make upthe eluent. The (D) phase is typically essentially pure organic solvent,but may, in certain embodiments be an organic solvent/water mixturecomprising mainly organic solvent. The (W) phase is typicallyessentially pure water, but may, in certain embodiments be an organicsolvent/water mixture comprising mainly water, for example a 98%water/2% methanol mixture.

A further illustration of option (1) is shown in FIG. 6. Here there isno separate water injection point, and instead an aqueous organicsolvent desorbent is injected at (D).

In option (1), the separation into raffinate and extract stream can beaided by varying the desorbing power of the eluent within eachchromatographic apparatus. This can be achieved by introducing theorganic solvent (or organic solvent rich) component of the eluent andthe water (or water rich) component at different points in eachchromatographic apparatus. Thus, typically, the organic solvent isintroduced upstream of the extract take-off point and the water isintroduced between the extract take-off point and the point ofintroduction of the feed into the chromatographic apparatus, relative tothe flow of eluent in the system. This is shown in FIG. 4.

Typically, in option (1), the aqueous organic solvent eluent used in thefirst SMB separation step contains more organic solvent than the eluentused in the second SMB separation step, i.e. the water:organic solventratio in the first SMB separation step is lower than the water:organicsolvent ratio in the second SMB separation step.

In option (1), the SMB separation can be aided by varying the rates atwhich liquid collected via the extract and raffinate streams in thefirst and second SMB separation steps is recycled back into thechromatographic apparatus used in that SMB separation step.

Typically, in option (1), the rate at which liquid collected via theextract stream in the first SMB separation step is recycled back intothe chromatographic apparatus used in the first SMB separation step isfaster than the rate at which liquid collected via the extract stream inthe second SMB separation step is recycled back into the chromatographicapparatus used in the second SMB separation step.

In option (1) the first raffinate stream in the first SMB separationstep is typically removed downstream of the point of introduction of theinput stream into the chromatographic apparatus used in the first SMBseparation step, with respect to the flow of eluent.

In option (1), the first extract stream in the first SMB separation stepis typically removed upstream of the point of introduction of the inputstream into the chromatographic apparatus used in the first SMBseparation step, with respect to the flow of eluent.

In option (1), the second raffinate stream in the second SMB separationstep is typically removed downstream of the point of introduction of thefirst product into the chromatographic apparatus used in the second SMBseparation step, with respect to the flow of eluent.

In option (1), the second extract stream in the second SMB separationstep is typically collected upstream of the point of introduction of thefirst product into the chromatographic apparatus used in the second SMBseparation step, with respect to the flow of eluent.

Typically in option (1), the organic solvent or aqueous organic solventis introduced into the chromatographic apparatus used in the first SMBseparation step upstream of the point of removal of the first extractstream, with respect to the flow of eluent.

Typically in option (1), when water is introduced into thechromatographic apparatus used in the first SMB separation step, thewater is introduced into the chromatographic apparatus used in the firstSMB separation step upstream of the point of introduction of the inputstream but downstream of the point of removal of the first extractstream, with respect to the flow of eluent.

Typically in option (1), the organic solvent or aqueous organic solventis introduced into the chromatographic apparatus used in the second SMBseparation step upstream of the point of removal of the second extractstream, with respect to the flow of eluent.

Typically in option (1), when water is introduced into thechromatographic apparatus used in the second SMB separation step, thewater is introduced into the chromatographic apparatus used in thesecond SMB separation step upstream of the point of introduction of thefirst product but downstream of the point of removal of the secondextract stream, with respect to the flow of eluent.

Option (2) is suitable for purifying DHA from an input stream.

Option (2) is illustrated in FIG. 3. An input stream F comprising thesecond product (B) and more polar (C) and less polar (A) components ispurified in the first SMB separation step. In the first SMB separationstep, the more polar components (C) are removed as raffinate stream R1.The second product (B) and less polar components (A) are collected asextract stream E1. Extract stream E1 is the first product which is thenpurified in the second SMB separation step. In the second SMB separationstep, the less polar components (A) are removed as extract stream E2.The second product (B) is collected as raffinate stream R2.

Option (2) is illustrated in more detail in FIG. 5. FIG. 5 is identicalto FIG. 3, except that the points of introduction of the organic solventdesorbent (D) and water (W) into each chromatographic apparatus areshown. As above, the (D) phase is typically essentially pure organicsolvent, but may, in certain embodiments be an organic solvent/watermixture comprising mainly organic solvent. The (W) phase is typicallyessentially pure water, but may, in certain embodiments be an organicsolvent/water mixture comprising mainly water, for example a 98%water/2% methanol mixture.

A further illustration of option (2) is shown in FIG. 7. Here there isno separate water injection point, and instead an aqueous organicsolvent desorbent is injected at (D).

Typically in option (2), the rate at which liquid collected via theraffinate stream in the first SMB separation step is reintroduced intothe chromatographic apparatus used in the first SMB separation step isfaster than the rate at which liquid collected via the raffinate streamin the second SMB separation step is reintroduced into thechromatographic apparatus used in the second SMB separation step.

Typically in option (2), the aqueous organic solvent eluent used in thefirst SMB separation step contains less organic solvent than the eluentused in the second SMB separation step, i.e. the water:organic solventratio in the first SMB separation step is higher than in the second SMBseparation step.

In option (2) the first raffinate stream in the first separation step istypically removed downstream of the point of introduction of the inputstream into the chromatographic apparatus used in the first SMBseparation step, with respect to the flow of eluent.

In option (2), the first extract stream in the first SMB separation stepis typically removed upstream of the point of introduction of the inputstream into the chromatographic apparatus used in the first SMBseparation step, with respect to the flow of eluent.

In option (2), the second raffinate stream in the second SMB separationstep is typically removed downstream of the point of introduction of thefirst product into the chromatographic apparatus used in the second SMBseparation step, with respect to the flow of eluent.

In option (2), the second extract stream in the second SMB separationstep is typically collected upstream of the point of introduction of thefirst product into the chromatographic apparatus used in the second SMBseparation step, with respect to the flow of eluent.

Typically in option (2), the organic solvent or aqueous organic solventis introduced into the chromatographic apparatus used in the first SMBseparation step upstream of the point of removal of the first extractstream, with respect to the flow of eluent.

Typically in option (2), when water is introduced into thechromatographic apparatus used in the first SMB separation step, thewater is introduced into the chromatographic apparatus used in the firstSMB separation step upstream of the point of introduction of the inputstream but downstream of the point of removal of the first extractstream, with respect to the flow of eluent.

Typically in option (2), the organic solvent or aqueous organic solventis introduced into the chromatographic apparatus used in the second SMBseparation step upstream of the point of removal of the second extractstream, with respect to the flow of eluent.

Typically in option (2), when water is introduced into thechromatographic apparatus used in the second SMB separation step, thewater is introduced into the chromatographic apparatus used in thesecond SMB separation step upstream of the point of introduction of thefirst product but downstream of the point of removal of the secondextract stream, with respect to the flow of eluent.

In this “back-to-back” SMB process, each of the simulated or actualmoving bed chromatography apparatus used in the first and second SMBseparation steps preferably consist of eight chromatographic columns.These are referred to as columns 1 to 8. In each apparatus the eightcolumns are arranged in series so that the bottom of column 1 is linkedto the top of column 2, the bottom of column 2 is linked to the top ofcolumn 3 . . . etc. . . . and the bottom of column 8 is linked to thetop of column 1. These linkages may optionally be via a holdingcontainer, with a recycle stream into the next column. The flow ofeluent through the system is from column 1 to column 2 to column 3 etc.The effective flow of adsorbent through the system is from column 8 tocolumn 7 to column 6 etc.

This is illustrated in FIG. 8. An input stream F comprising the secondproduct (B) and more polar (C) and less polar (A) components isintroduced into the top of column 5 in the chromatographic apparatusused in the first SMB separation step. Organic solvent desorbent isintroduced into the top of column 1 of the chromatographic apparatusused in the first SMB separation step. Water is introduced into the topof column 4 of the chromatographic apparatus used in the first SMBseparation step. In the first SMB separation step, the less polarcomponents (A) are removed as extract stream E1 from the bottom ofcolumn 2. The second product (B) and more polar components (C) areremoved as raffinate stream R1 from the bottom of column 7. Raffinatestream R1 is the first product which is then purified in the second SMBseparation step, by being introduced into the chromatographic apparatusused in the second SMB separation step at the top of column 5. Organicsolvent desorbent is introduced into the top of column 1 in thechromatographic apparatus used in the second SMB separation step. Wateris introduced into the top of column 4 in the chromatographic apparatusused in the second SMB separation step. In the second SMB separationstep, the more polar components (C) are removed as raffinate stream R2at the bottom of column 7. The second product (B) is collected asextract stream E2 at the bottom of column 2.

In the “back-to-back” SMB process shown in FIG. 8, organic solvent istypically introduced into the top of column 1 of the chromatographicapparatus used in the first SMB separation step.

In the “back-to-back” SMB process shown in FIG. 8, water is typicallyintroduced into the top of column 4 of the chromatographic apparatusused in the first SMB separation step.

In the “back-to-back” SMB process shown in FIG. 8, organic solvent istypically introduced into the top of column 1 of the chromatographicapparatus used in the second SMB separation step.

In the “back-to-back” SMB process shown in FIG. 8, organic solvent istypically introduced into the top of column 4 of the chromatographicapparatus used in the second SMB separation step.

In the “back-to-back” SMB process shown in FIG. 8, the input stream istypically introduced into the top of column 5 of the chromatographicapparatus used in the first SMB separation step.

In the “back-to-back” SMB process shown in FIG. 8, a first raffinatestream is typically collected as the first product from the bottom ofcolumn 7 of the chromatographic apparatus used in the first SMBseparation step. This first product is then purified in the second SMBseparation step and is typically introduced into the top of column 5 ofthe chromatographic apparatus used in the second SMB separation step.The first raffinate stream may optionally be collected in a containerbefore being purified in the second SMB separation step.

In the “back-to-back” SMB process shown in FIG. 8, a first extractstream is typically removed from the bottom of column 2 of thechromatographic apparatus used in the first SMB separation step. Thefirst extract stream may optionally be collected in a container andreintroduced into the top of column 3 of the chromatographic apparatusused in the first SMB separation step.

In the “back-to-back” SMB process shown in FIG. 8, a second raffinatestream is typically removed from the bottom of column 7 of thechromatographic apparatus used in the second SMB separation step.

In the “back-to-back” SMB process shown in FIG. 8, a second extractstream is typically collected from the bottom of column 2 of thechromatographic apparatus used in the second SMB separation step. Thissecond extract stream typically contains the second product. The secondextract stream may optionally be collected in a container andreintroduced into the top of column 3 of the chromatographic apparatusused in the second SMB separation step.

In the “back-to-back” SMB process shown in FIG. 8, the eluent used istypically as defined above.

Typically, in this “back-to-back” SMB process, the water:organic solventratio in the chromatographic apparatus used in the first SMB separationstep is lower than the water:organic solvent ratio in thechromatographic apparatus used in the second SMB separation step. Thus,the eluent in the first SMB separation step typically contains moreorganic solvent than the eluent used in the second SMB separation step.

In this “back-to-back” SMB process, the water:organic solvent ratio inthe first SMB separation step is typically from 0.5:99.5 to 1.5:98.5parts by volume. The water:organic solvent ratio in the second SMBseparation step is typically from 2:98 to 6:94 parts by volume.

In this “back-to-back” SMB process, although the apparatus of FIG. 8 isconfigured as shown in FIG. 10a , the configurations shown in FIGS. 10band 10c could also be used.

This “back-to-back” SMB process is also illustrated in FIG. 9. An inputstream F comprising the second product (B) and more polar (C) and lesspolar (A) components is introduced into the top of column 5 in thechromatographic apparatus used in the first SMB separation step. Aqueousorganic solvent desorbent is introduced into the top of column 1 in thechromatographic apparatus used in the first SMB separation step. In thefirst SMB separation step, the less polar components (A) are removed asextract stream E1 from the bottom of column 2. The second product (B)and more polar components (C) are removed as raffinate stream R1 fromthe bottom of column 7. Raffinate stream R1 is the first product whichis purified in the second SMB separation step by being introduced intothe top of column 4 of the chromatographic apparatus used in the secondSMB separation step. Aqueous organic solvent desorbent is introducedinto the top of column 1 in the chromatographic apparatus used in thesecond SMB separation step. In the second SMB separation step, the morepolar components (C) are removed as raffinate stream R2 at the bottom ofcolumn 7. The second product (B) is collected as extract stream E2 atthe bottom of column 2.

In the “back-to-back” SMB process shown in FIG. 9, aqueous organicsolvent is typically introduced into the top of column 1 in thechromatographic apparatus used in the first SMB separation step.

In the “back-to-back” SMB process shown in FIG. 9, aqueous organicsolvent is typically introduced into the top of column 9 in thechromatographic apparatus used in the second SMB separation step.

In the “back-to-back” SMB process shown in FIG. 9, the input stream istypically introduced into the top of column 5 in the chromatographicapparatus used in the first SMB separation step.

In the “back-to-back” SMB process shown in FIG. 9, a first raffinatestream is typically collected as the first product from the bottom ofcolumn 7 of the chromatographic apparatus used in the first SMBseparation step. This first product is then purified in the second SMBseparation step and is typically introduced into the top of column 5 ofthe chromatographic apparatus used in the second SMB separation step.The first raffinate stream may optionally be collected in a containerbefore being purified in the second SMB separation step.

In the “back-to-back” SMB process shown in FIG. 9, a first extractstream is typically removed from the bottom of column 2 of thechromatographic apparatus used in the first SMB separation step. Thefirst extract stream may optionally be collected in a container and aportion reintroduced into the top of column 3 of the chromatographicapparatus used in the first SMB separation step. The rate of recycle ofliquid collected via the extract stream in the first SMB separation stepback into the chromatographic apparatus used in the first SMB separationstep is the rate at which liquid is pumped from this container into thetop of column 3.

In the “back-to-back” SMB process shown in FIG. 9, a second raffinatestream is typically removed from the bottom of column 7 of thechromatographic apparatus used in the first SMB separation step.

In the “back-to-back” SMB process shown in FIG. 9, a second extractstream is typically collected from the bottom of column 2 of thechromatographic apparatus used in the first SMB separation step. Thissecond extract stream typically contains the second product. The secondextract stream may optionally be collected in a container and a portionreintroduced into the top of column 3 of the chromatographic apparatusused in the first SMB separation step. The rate of recycle of liquidcollected via the extract stream from the second SMB separation stepback into the chromatographic apparatus used in the second SMBseparation step is the rate at which liquid is pumped from thiscontainer into the top of column 3.

In the “back-to-back” SMB process shown in FIG. 9, the eluent used is asdefined above.

Typically, in this “back-to-back” SMB process, the water:organic solventratio in the chromatographic apparatus used in the first SMB separationstep is lower than the water:organic solvent ratio in thechromatographic apparatus used in the second SMB separation step. Thus,the eluent used in the first SMB separation step typically contains moreorganic solvent than the eluent used in the second SMB separation step.

In this “back-to-back” SMB process, the water:organic solvent ratio inthe first SMB separation step is typically from 0.5:99.5 to 1.5:98.5parts by volume. The water:organic solvent ratio in the second SMBseparation step is typically from 2:98 to 6:94 parts by volume.

In this “back-to-back” SMB process, the rate at which liquid collectedvia the extract stream from the first SMB separation step is recycledback into the chromatographic apparatus used in the first SMB separationstep is typically faster than the rate at which liquid collected via theextract stream from the second SMB separation step is recycled back intothe chromatographic apparatus used in the second SMB separation step. Inthis case, the aqueous organic solvent eluent is typically substantiallythe same in each SMB separation step.

In this “back-to-back” SMB process, although the apparatus of FIG. 9 isconfigured as shown in FIG. 10a , the configurations shown in FIGS. 10band 10c could also be used.

Typically, at least one of the first and second chromatographicseparation steps involve at least one, for example one, “back-to-back”SMB process as defined above.

Typically, the PUFA product is separated from different components ofthe feed mixture in each chromatographic separation step.

Typically, the PUFA product is separated from one or more of the C18fatty acid impurities disclosed above in the first and/or secondseparation steps. Typically the PUFA product is separated from one ormore of the C18 fatty acid impurities discussed above in only one of thefirst and second separation steps.

More typically, the PUFA product is separated from ALA, ALA mono-, di-and triglycerides and ALA C₁-C₄ alkyl esters in the first and/or secondseparation steps.

More typically, the PUFA product is separated from GLA, GLA mono-, di-and triglycerides and GLA C₁-C₄ alkyl esters in the first and/or secondseparation steps.

Preferably, the PUFA product is separated from C18 fatty acids, C18fatty acid mono-, di- and triglycerides and C18 fatty acid alkyl estersin the first and/or second separation steps.

Typically, the intermediate product has a lower concentration of one ormore of the C18 fatty acid impurities disclosed above than the feedmixture; and/or the PUFA product produced in the second separation stephas a lower concentration of one or more of the C18 fatty acidimpurities disclosed above than the intermediate product.

More typically, the intermediate product has a lower concentration ofimpurities selected from ALA, mono, di- and triglycerides of ALA andC₁-C₄ alkyl esters of ALA than the feed mixture; and/or the PUFA productproduced in the second separation step has a lower concentration of saidimpurities than the intermediate product.

More typically, the intermediate product has a lower concentration ofimpurities selected from GLA, mono, di- and triglycerides of GLA andC₁-C₄ alkyl esters of GLA than the feed mixture; and/or the PUFA productproduced in the second separation step has a lower concentration of saidimpurities than the intermediate product.

Preferably, the intermediate product has a lower concentration of C18fatty acids or C18 fatty acid derivatives than the feed mixture; or thePUFA product produced in the second separation step has a lowerconcentration of C18 fatty acids or C18 fatty acid derivatives than theintermediate product. In certain embodiments the intermediate producthas a lower concentration of C18 fatty acids or C18 fatty acidderivatives than the feed mixture; and the PUFA product produced in thesecond separation step has a lower concentration of C18 fatty acids orC18 fatty acid derivatives than the intermediate product.

A lower concentration typically means a concentration which is lower byan amount of 5 wt % or more, more typically 10 wt % or more, preferably20 wt % or more, more preferably 30 wt % or more, even more preferably40 wt % or more, yet more preferably 50 wt % or more, yet morepreferably 60 wt % or more, yet more preferably 70 wt % or more, yetmore preferably 80 wt % or more, yet more preferably 90 wt % or more.Thus, when the intermediate product has a lower concentration of one ormore of the C18 fatty acid impurities disclosed above than the feedmixture, the concentration of the C18 fatty acid impurities in theintermediate product is typically 10 wt % or more, preferably 20 wt % ormore etc, lower than the concentration of the C18 fatty acid impuritiesin the feed mixture. When the PUFA product produced in the secondseparation step has a lower concentration of one or more of the C18fatty acid impurities disclosed above than the intermediate product, theconcentration of the C18 fatty acid impurities in the PUFA product istypically 10 wt % or more, preferably 20 wt % or more etc, lower thanthe concentration of the C18 fatty acid impurities in the intermediateproduct.

Typically, the first organic solvent is acetonitrile, and theintermediate product has a lower concentration of one or more of the C18fatty acid impurities disclosed above than the feed mixture.Alternatively, the second organic solvent is acetonitrile, and the PUFAproduct produced in the second separation step has a lower concentrationof one or more of the C18 fatty acid impurities disclosed above than theintermediate product.

Preferably, the PUFA product is EPA ethyl ester, and (i) the firstorganic solvent is acetonitrile, and the intermediate product has alower concentration of one or more of the C18 fatty acid impuritiesdisclosed above than the feed mixture, or (ii) the second organicsolvent is acetonitrile, and the PUFA product produced in the secondseparation step has a lower concentration of one or more of the C18fatty acid impurities disclosed above than the intermediate product.

More preferably, the PUFA product is EPA ethyl ester, and (i) the firstorganic solvent is acetonitrile, the second organic solvent is methanoland the intermediate product has a lower concentration of one or more ofthe C18 fatty acid impurities disclosed above than the feed mixture, or(ii) the first organic solvent is methanol, the second organic solventis acetonitrile, and the PUFA product produced in the second separationstep has a lower concentration of one or more of the C18 fatty acidimpurities disclosed above than the intermediate product.

More preferably, the PUFA product is EPA ethyl ester, and (i) the firstorganic solvent is acetonitrile, the second organic solvent is methanol,and the first chromatographic separation step comprises introducing thefeed mixture into a stationary bed apparatus and the secondchromatographic separation step comprises introducing the intermediateproduct into a simulated or actual moving bed chromatography apparatus;or (ii) the first organic solvent is methanol and the second organicsolvent is acetonitrile, the first chromatographic separation stepcomprises introducing the feed mixture into a simulated or actual movingbed chromatography apparatus and the second chromatographic separationstep comprises introducing the intermediate product into a stationarybed chromatography apparatus.

Even more preferably, the PUFA product is EPA ethyl ester, and (i) thefirst organic solvent is acetonitrile, the second organic solvent ismethanol, the intermediate product has a lower concentration of one ormore of the C18 fatty acid impurities disclosed above than the feedmixture, and the first chromatographic separation step comprisesintroducing the feed mixture into a stationary bed apparatus and thesecond chromatographic separation step comprises introducing theintermediate product into a simulated or actual moving bedchromatography apparatus; or (ii) the first organic solvent is methanol,the second organic solvent is acetonitrile, the PUFA product produced inthe second separation step has a lower concentration of one or more ofthe C18 fatty acid impurities disclosed above than the intermediateproduct, and the first chromatographic separation step comprisesintroducing the feed mixture into a simulated or actual moving bedchromatography apparatus and the second chromatographic separation stepcomprises introducing the intermediate product into a stationary bedchromatography apparatus.

The present invention also provides a PUFA product, as defined above,which is obtainable by the process of the present invention.

The present invention also provides a composition comprising a PUFAproduct of the present invention.

Such compositions typically contain, as PUFA product, EPA or EPA ethylester.

The PUFA product is typically present in the compositions in an amountin an amount greater than 90 wt %, preferably greater than 95 wt %, morepreferably greater than 97 wt %, even more preferably greater than 98 wt%, still more preferably greater than 98.4 wt %.

Preferably, the PUFA product is EPA or EPA ethyl ester and is present inthe compositions in an amount in an amount greater than 90 wt %,preferably greater than 95 wt %, more preferably greater than 97 wt %,even more preferably greater than 98 wt %, still more preferably greaterthan 98.4 wt %, for example in an amount between 98 and 99.5 wt %.

Typically, the PUFA product contains less than 1 wt % of one or more ofthe C18 fatty acid impurities disclosed above.

Typically, the PUFA product contains less than 1 wt % of alpha-linolenicacid (ALA), ALA mono-, di- and triglyceride and ALA C₁-C₄ alkyl esterimpurities. More typically, the PUFA product contains less than 1 wt %of impurities which are ALA and derivatives thereof. Typical ALAderivatives are as defined above for PUFA derivatives.

Typically, the PUFA product contains less than 1 wt % of gamma-linolenicacid (GLA), GLA mono-, di- and triglyceride and GLA C₁-C₄ alkyl esterimpurities. More typically, the PUFA product contains less than 1 wt %of impurities which are GLA and derivatives thereof. Typical GLAderivatives are as defined above for PUFA derivatives.

Typically, the PUFA product contains less than 1 wt % of C18 fatty acidimpurities, C18 fatty acid mono-, di- and triglyceride and C18 fattyacid alkyl ester impurities. More typically, the PUFA product containsless than 1 wt % of impurities which are C18 fatty acids and derivativesthereof. For the avoidance of doubt, in this embodiment the maximumamount of all such impurities is 1 wt %. Typical C18 fatty acidderivatives are as defined above for PUFA derivatives. As used herein, aC18 fatty acid is a C18 aliphatic monocarboxylic acid having a straightor branched hydrocarbon chain. Typical C18 fatty acids include stearicacid (C18:0), oleic acid (C18:1n9), vaccenic acid (C18:1n7), linoleicacid (C18:2n6), gamma-linolenic acid/GLA (C18:3n6), alpha-linolenicacid/ALA (C18:3n3) and stearidonic acid/SDA (C18:4n3).

As explained above, typically the amount of the above-mentionedimpurities in the PUFA product is less than 1 wt %. Preferably, theamount of the above-mentioned impurities is less than 0.5 wt %, morepreferably less than 0.25 wt %, even more preferably less than 0.1 wt %,yet more preferably less than 0.05 wt %, yet more preferably less than0.01 wt %, yet more preferably less than 0.001 wt %, yet more preferablyless than 0.0001 wt %, yet more preferably less than 0.00001 wt %.

In certain preferred embodiments, the PUFA product is substantially freeof the above-mentioned impurities.

The PUFA product is not ALA, GLA, linoleic acid, an ALA mono- di- ortriglyceride, a GLA mono- di- or triglyceride, an oleic acid mono, di-or triglyceride, an ALA C₁-C₄ alkyl ester, a GLA C₁-C₄ alkyl ester or anoleic acid C₁-C₄ alkyl ester or a mixture thereof. Typically, the PUFAproduct is not ALA, GLA, linoleic acid, or a derivative or mixturesthereof. Typical ALA, GLA and linoleic acid derivatives are as definedabove for PUFA derivatives.

Typically, the PUFA product is not a C18 PUFA, a C18 PUFA mono-, di- ortriglyceride, or a C18 PUFA alkyl ester. More typically, the PUFAproduct is not a C18 PUFA or a C18 PUFA derivative. Typical C18 PUFAsinclude linoleic acid (C18:2n6), GLA (C18:3n6), and ALA (C18:3n3).

Typically, the composition comprises, as PUFA product, EPA or EPA ethylester present in an amount between 98 and 99.5 wt %, the compositioncontaining less than 1 wt % of ALA ethyl ester.

Typically, the composition comprises, as PUFA product, EPA or EPA ethylester present in an amount between 98 and 99.5 wt %, the compositioncontaining less than 1 wt % of GLA ethyl ester.

Preferably, the composition comprises, as PUFA product, EPA or EPA ethylester present in an amount between 98 and 99.5 wt %, the compositioncontaining less than 1 wt % of ALA, ALA mono-, di- and triglycerides andALA C₁-C₄ alkyl esters.

Preferably, the composition comprises, as PUFA product, EPA or EPA ethylester present in an amount between 98 and 99.5 wt %, the compositioncontaining less than 1 wt % of GLA, GLA mono-, di- or triglycerides andGLA C₁-C₄ alkyl esters.

More preferably, the composition comprises, as PUFA product, EPA ethylester present in an amount between 98 and 99.5 wt %, the compositioncontaining less than 1 wt % of ALA, ALA mono-, di- or triglycerides andALA C₁-C₄ alkyl esters.

More preferably, the composition comprises, as PUFA product, EPA ethylester present in an amount between 98 and 99.5 wt %, the compositioncontaining less than 1 wt % of GLA, GLA mono-, di- or triglycerides andGLA C₁-C₄ alkyl esters.

The following Examples illustrate the invention.

EXAMPLES Example 1 First Chromatographic Separation Step

A fish oil derived feedstock (55 weight % EPA ethyl ester (EE), 5 weight% DHA EE) with fatty acid profile as shown in FIG. 16 was fractionatedusing an actual moving bed chromatography system using bonded C18 silicagel (particle size 300 μm, particle porosity 150 angstroms) asstationary phase and aqueous methanol (typically 0.5% to 10% water) aseluent through a “single pass” SMB apparatus consisting of 15 columns(diameter: 76.29 mm, length: 914.40 mm) connected in series.

The operating parameters and flowrates are as follows.

(typical flow scheme as per FIG. 8)

Step time: 750 secs

Cycle time: 200 mins

Feed mixture feed rate (F1): 74 ml/min

Desorbent feed rate (D1): 6250 ml/min

Extract accumulation rate (E1): 1250 ml/min

Extract recycle rate (D1−E1): 5000 ml/min

Raffinate accumulation rate (R1): 1688 ml/min

Cycle time: 600 secs

The intermediate product produced by this process has a GC-FAMES traceas shown in FIG. 12. EPA EE is contained at 96.5% purity. The majorimpurity is ethyl-alpha linolenoate (ALA-C18:3n3) present at 0.9%. ALAis present in the raw material at 0.65%. ALA can therefore be seen toco-elute with EPA using methanol/water as the mobile phase.Methanol/water is, however, very efficient at removing the closelyrelated component ethyl-docosahexaenoate (DHA-C22:6n3).

Second Chromatographic Separation Step

The intermediate product produced in the first chromatographicseparation step was further purified by preparative HPLC in a fixed bedusing an acetonitrile/water mobile phase mix. Acetonitrile/water in aratio of 87:13 by wt was utilised. An HPLC column of dimensions 600mm×900 mm packed with c18 bonded silica (20 μm particle size) is usedwith a feed mixture injection volume of 1400 ml and a desorbent flowrate of 2200 ml/min.

The final PUFA product produced was analysed by GC FAMES and the traceis shown in FIG. 13. It can be see that ALA has been completely removedand the EPA purity increased to 98.5%.

Alternative Second Chromatographic Separation Step

The intermediate product produced in the first chromatographicseparation step was fractionated using an actual moving bedchromatography system using bonded C18 silica gel (particle size 300 μm,particle porosity 150 angstroms) as stationary phase and aqueousacetonitrile (12% water) as eluent through a “single pass” SMB apparatusconsisting of 8 columns (diameter: 76.29 mm, length: 914.40 mm)connected in series.

The operating parameters and flowrates are as follows.

(typical flow scheme as per FIG. 8)

Step time: 780 secs

Feed mixture feed rate (F1): 90 ml/min

Desorbent feed rate (D1): 6500 ml/min

Extract accumulation rate (E1): 1400 ml/min

Extract recycle rate (D1−E1): 5100 ml/min

Raffinate accumulation rate (R1): 1690 ml/min

Cycle time: 600 secs

Example 2 First Chromatographic Separation Step

A fish oil derived feedstock (55 weight % EPA EE, 5 weight % DHA EE)with fatty acid profile as shown in FIG. 16 was subjected to preparativeHPLC separation using an acetonitrile/water eluent. The mobile phaseused is 87:13 Acetonitrile:water. An HPLC column of dimensions 600mm×900 mm packed with c18 bonded silica (20 μm particle size) is usedwith a feed mixture injection volume of 600 ml and a desorbent flow rateof 2200 ml/min. The intermediate product produced was analysed by GCFAME and the trace is shown as FIG. 14.

It can be seen that ethyl-alpha-linolenoate (ALA-C18:3n3) was completelyremoved from the feed mixture. However a purity level of only 92.5% EPAEE was achieved mainly due to the presence of a high level ofethyl-docosahexaenoate (DHA-C22:6n3).

Alternative First Chromatographic Separation Step

A fish oil derived feedstock (55 weight % EPA EE, 5 weight % DHA EE)with fatty acid profile as shown in FIG. 16 was fractionated using anactual moving bed chromatography system using bonded C18 silica gel(particle size 300 μm, particle porosity 150 angstroms) as stationaryphase and aqueous acetonitrile (typically 4% to 18% water) as eluentthrough a “single pass” SMB apparatus consisting of 15 columns(diameter: 76.29 mm, length: 914.40 mm) connected in series.

The operating parameters and flowrates are as follows.

(typical flow scheme as per FIG. 8)

Step time: 600 secs

Feedstock (F) feed rate: 105 ml/min

Desorbent (D) feed rate: 4800 ml/min

Extract rate: 1250 ml/min

Raffinate rate: 1800 ml/min

Second Chromatographic Separation Step

The intermediate product produced was subjected to further purificationusing preparative HPLC using as eluent methanol/water at 88:12 ratio bywt. An HPLC column of dimensions 600 mm×900 mm packed with c18 bondedsilica (20 μm particle size) is used with a feed mixture injectionvolume of 1250 ml and a desorbent flow rate of 2200 ml/min.

The final product produced has a GC FAMES trace as shown in FIG. 15. Theproduct produced contains EPA EE at 99% purity.

Thus, it can be seen that the outcome from performing acetonitrile/waterseparation first followed by methanol/water is essentially the same asperforming methanol/water first followed by acetonitrile/water. In eachcase combining a step involving methanol/water and a further stepinvolving acetonitrile/water is advantageous in preparing a highlypurified EPA (EE) concentrate at .about.99% purity with a low content ofC18 fatty acid impurities, for example ALA.

Alternative Second Chromatographic Separation Step

The intermediate product produced was fractionated using an actualmoving bed chromatography system using bonded C18 silica gel (particlesize 300 μm, particle porosity 150 angstroms) as stationary phase andaqueous methanol (7% water) as eluent through a “single pass” SMBconsisting of 8 columns (diameter: 76.29 mm, length: 914.40 mm)connected in series.

The operating parameters and flowrates are as follows.

(typical flow scheme as per FIG. 8)

Step time: 960 secs

Feedstock (F) feed rate: 45 ml/min

Desorbent (D) feed rate: 3975 ml/min

Extract rate: 3655 ml/min

Raffinate rate: 2395 ml/min

Comparative Example 1

A fish oil derived feedstock (55 weight % EPA EE, 5 weight % DHA EE)with fatty acid profile as shown in FIG. 16 is fractionated in first andsecond chromatographic separation steps using an actual moving bedchromatography system using bonded C18 silica gel (particle size 300 μm,particle porosity 150 angstroms) as stationary phase and aqueousmethanol as eluent in both separation steps.

First separation step performed on a series of 8 columns (diameter:76.29 mm, length: 914.40 mm) connected in series.

The operating parameters and flowrates are as follows.

(typical flow scheme as per FIG. 8)

Feed mixture feed rate (F1): 34 ml/min

Desorbent feed rate (D1): 14438 ml/min

Extract accumulation rate (E1): 9313 ml/min

Extract recycle rate (D1−E1): 5125 ml/min

Raffinate accumulation rate (R1): 1688 ml/min

Cycle time: 1200 secs

Second separation step performed on a second series of 7 columns(diameter: 76.29 mm, length: 914.40 mm) connected in series.

Second intermediate product feed rate (F3): 40 ml/min

Desorbent feed rate (D3): 6189 ml/min

Extract accumulation rate (E3): 1438 ml/min

Extract recycle rate (D3−E3): 4750 ml/min

Raffinate accumulation rate (R3): 1438 ml/min

Cycle time: 1080 secs

The comparative example produced an EPA concentrate with a lessadvantageous impurity profile. The upper purity achievable is limited inparticular by the presence of C18:3 components (GLA and ALA).

What is claimed is:
 1. A chromatographic separation process forrecovering a polyunsaturated fatty acid (PUFA) product from a feedmixture, which comprises: (a) purifying the feed mixture in a firstchromatographic separation step using as eluent a mixture of water and afirst organic solvent, to obtain an intermediate product; and (b)purifying the intermediate product in a second chromatographicseparation step using as eluent a mixture of water and a second organicsolvent, to obtain the PUFA product, wherein the second organic solventis different from the first organic solvent, and wherein the firstchromatographic separation step comprises introducing the feed mixtureinto a simulated or actual moving bed chromatography apparatus and thesecond chromatographic separation step comprises introducing theintermediate product into a stationary bed chromatography apparatus. 2.The process according to claim 1, wherein the first and second organicsolvents are chosen from alcohols, ethers, esters, ketones and nitriles.3. The process according to claim 2, wherein the ketone is acetone,methylethylketone or methyl isobutyl ketone (MIBK), preferably acetone.4. The process according to claim 1, wherein one of the first and secondorganic solvents is methanol.
 5. The process according to claim 1,wherein the second organic solvent is methanol.
 6. The process accordingto claim 1, wherein the first organic solvent:water ratio is from99.9:0.1 to 75:25 parts by volume.
 7. The process according to claim 6,wherein the first organic solvent:water ratio is from 99.5:0.5 to 80:20parts by volume.
 8. The process according to claim 1, wherein the secondorganic solvent:water ratio is from 99.9:0.1 to 75:25 parts by volume.9. The process according to claim 8, wherein the second organicsolvent:water ratio is from 90:10 to 85:15 parts by volume.
 10. Theprocess according to any claim 1, wherein the second organic solvent ismethanol, and the methanol:water ratio is from 95:5 to 85:15 parts byvolume.
 11. The process according to any claim 10, wherein the secondorganic solvent is methanol, and the methanol:water ratio is from 93:7to 90:10 parts by volume.
 12. The process according to claim 1, whereinthe PUFA 5 product is at least one ω-3 PUFA or at least one ω-3 PUFAderivative.
 13. The process according to claim 1, wherein the PUFAproduct is eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), EPAtriglyceride, DHA triglyceride, EPA ethyl ester or DHA ethyl ester. 14.The process according to claim 1, wherein the PUFA product is EPA, orEPA ethyl ester.
 15. The process according to claim 1, wherein the PUFAproduct is obtained in the second separation step at a purity greaterthan 95 wt %, preferably greater than 97 wt %, more preferably greaterthan 98 wt %, still more preferably greater than 98.4 wt %.
 16. Theprocess according to claim 1, wherein the first organic solvent isacetone, and the second organic solvent is methanol.
 17. A PUFA productobtainable by the process of claim
 1. 18. A composition comprising aPUFA product obtainable by the process of claim 1.